p . 1 B05134 6 

### 9 . Reference s [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 li ability of ~ the study result s

None 10 .2 Deviations from the protocol None

L~ 1A 1?1p . 2 B05134 6 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 (mmn ber of colonies/plate ) activa ti on concen trati on Base-pair change ty e Frames hiR type sy ste m (p~ late) TA 100 TA 1535 WP2 uvr A - TA 98 TA 1537 110 10 21 16 14 Solvent control 107 109 13 12 17 19 15 16 12 1 3

101 11 21 11 15 0 .305 107 104 7 9 15 18 12 12 15 15

96 5 23 9 1 7 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 1 5 85 10 22 11 13 (-) 19 .5 103 94 10 10 23 23 15 13 9 11 110 11 20 16 1 7 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 1 4

108 10 19 11 13 1250 99 104 6 8 14 17 12 12 14 14

112 11 23 14 1 9 5000 83 98 11 11 19 21 14 14 16 18

84 11 21 16 15 Solvent cont ro l 92 88 8 10 22 22 21 19 12 1 4

117 8 23 10 17 0 .305 106 112 8 8 18 21 13 12 18 18

89 13 27 19 1 9 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 1 7 103 8 20 20 15 (+) 19 .5 90 97 5 7 21 21 17 19 13 14 83 7 22 18 1 4 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 1 1 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 53 0 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 10 8 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 concen tr ati ons of the test subst ance . Z `i . '

~ ~~p . 3


### B051346 Table 2 Results of the Main Tes t Test substance : MTDID366 3 Testing periods From April 25, 2006 to April 28, 2006 Metaboli c Test material Number of revertants (number of colonies/plate ) activati on concen tra ti on Base-pair change type Frameshift typ e 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 s9mb x 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 ( 1 2

100 6 20 26 11 313 76 ( 88 ) 9 8 28 ( 24 ) 21 ( 24 ) 11 ( 11 )

80 7 27 20 1 5 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 1 1 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 con trol (Awplate ) not requiring Number of 807 602 669 482 33 3 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 -A A ~ Posi ti ve concentration 1 2 10 0 .5 2 control (49/plate) I re quiring Number of 787 238 756 437 13 2 S9 mix revertant/plat e late 822 ( 805 ) 256 ( 247 ) 881 ( 819 ) 422 ( 430 ) 112 ( 122 AF-2 : 2-(2-fiuyl)-3-(5-nitro-2-fv rylJacry 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 of revertants" - the observed value and average value in order beginning with the low concentrations of the test substance .

GJ ' 

### ~ Sbp . 4 B05134 6 (TA100~ 200 200 .~- -S9mix ••-•© . . . . . +S9mi x

150 a) 150
~ CO CO

~ 'ay ~ W 0 ° 100 100 0 ° °

- 0 +~ - ~ `c 4~ L CD C D Q> ° D 50 50

~ 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 20 2 0 O U o N U ~ C i CO C D 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)

^~- 

# 1 IA~

### p . 5 B051346 ~ WP2 uvrA- )

60 40

--~ . .-- -S9mix -•© . •••• +S9mix 30 a) 4 5
4~ cC lC Q Q N 0) y U C ° 30 0 201 - ~

- -O- - - O - " 0 0 k<%I ;v 4- 1 c _ ~ © c cc 4.j CD m 15 1 0 \ 0

0 0 313 625 1250 2500 5000 0 0 313 625 1250 2500 500 0

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 3 0 (D CD R ~ a n CO CO 3 . ' . . ~ a> c `©_ _ •

0 20 -°0 2 0 0 C i* .+ . ~ w ~ ~ U 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 4 0 --f-- -S9mix ••• . © . . . . . +S9mix
30 30 ~ 41 CO c 4 C L m co w y 0 20 -o° 2 0 U U 4a 4-J ++ .{-~i CD CD ~ ~ 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 v alue -S9mix +S9mix Strain Average Range No . of Average Range No . of assay assa y 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 6 6 ~ TA98 17 8-26 67 21 11-31 67

TA1537 8 3-14 66 11 5-16 66 2 . Background data of the positive con tr ol valu e -S9mix +S9mix Strain Average Range No . of Average Range No . of assay assa y 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 3 4 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 Statemen t Title : Bacte rial Reverse Mutation Test of MTDID3663 Study No . : B05134 6 By the inspections and audits listed below, I hereby ce rtify that the study was car ri ed out in accord an ce with its protocol and with our Standard Operating Procedures, and that the report faithfully describes the methods and procedures applied, and that the repo rted results accurately reflect the raw data of the study . St andard Concerning Testing Facilities Implementing studies Related to New Chemical Substances, (Pharmaceutic al and Food Safety Bureau, M anufacturing Industri es Bureau, ~ Environment al Policy Bureau, Ministry of the Environment, Pharmaceutic al and Foo d

affairs No . 1121003, November 17, 2003, No .3, November 21, 2003, Minist ry of Health, Labor, and Welfare, Ministry of Economy, Trade, and Industry , No .031121004) Noti fication for Stan dard of Testing Facilities Based on Rule for Indust ri al Safety and Health, Article 34-3- 2 (Notifi cation No 76, Ministry of Labor, September 1, 1988 ) A Pa rt Revision of Notification for St andard of Testing Facilities Based on Rule for Industrial Safety and He alth, Article 34(3)- 2 (Notification No 13, Ministry of Labor, March 29, 2000) Date of repo rting to Inspection or audit Date of inspection the study directo r or audit and to the m anagement Study Protocol Ap ril 5, 2006 April 5, 200 6 Study Procedur e Dose-finding test, Pre-incubation April 18, 2006 Ap ri l 19, 2006 Dose-finding test, Colony count Ap ril 20, 2006 Ap ril 24, 2006 Main test, Pre-incubation April 26, 2006 Ap ril 27, 2006 Main test, Colony count April 28, 2006 Ap ril 28, 2006 Dra ft Repo rt June 2, 2006 June 2, 2006 Study Report June 8, 2006 June 8, 200 6 Quality Assur ance manager : Date : June 8, 2006 Hidetsugu Watabe se al ed General Testing Research Institute of Jap an Oilstuff Inspectors' Corporation -3,9- 

# ~SS

## FINAL REPORT M .S .I . Report No . A05039 7 Acute Toxicity Test of MTDID3663 ~ with Medaka ( Oryzias Wipes)

Submi

### tted to : Sumitomo 3M Limited

Prepared by : Mitsubishi Chemical Safety Institute Ltd . (M .S .I .)

August 31, 200 6

---~- , 

## ~ 5 lp

### Ao S09 7 Acute Toxicity Test of MTDID366 3 with Medaka ( O r

yzias latipes) (English version) Sponsor Sumitomo 3M Limite d Study Title Acute Toxicity Test of MTDID3663 with Medaka ( Oryzias latipes) Study No . A05039 7 This report is the English version of the original, which was wri tt en 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

~-- -

~

~~~AVJ rJa l 

## Compliance with the GLP Standard s

Yokohama Laborator

y Mitsubishi Chemical Safety Institute Ltd. Sponsor Sumitomo 3M Limite d Study Title Acute Toxicity Test of MTDID3663 with Medaka (Oryzias latipes) Study No . A05039 7 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 Directo r Midori MINO, B .Sc . Sealed date : August 31, 2006

~---

.~-3--- ~-}

### Quality Assurance Statement Yokohama Laborator

y Mitsubishi Chemical Safety Institute Ltd . Sponsor Sumitomo 3M Limite d Study Title Acute Toxicity Test of MTDID3663 with Medaka ( O ryzias latipes) Study No . A05039 7 We hereby ce rt ify that this study was carried out in accord an ce with its protocol and with our standard operating procedures . We certify that this repo rt faithfully describes the methods and procedures employed, and that the repo rted results reflect the raw data of the study, accurately . Date of Repo rting to Date of Inspection Management and Study Directo r 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 Observ ation of fish : August 1 1 , 2006 August 11, 200 6 

### Final repo rt : 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 . Purpos e 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 Guideline s 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 . Sponso r Sumitomo 3M Limite d 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, Japa n 7 . Test Facility (Laboratory) Yokohama Laborator y Mitsubishi Chemical Safety Institute Ltd . 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, Japan 8 . Study Directo r Midori MINO, B .Sc . Ecological Chemistry Division .-3-- ~(0d

### A0MJ47

9 . Personnel in Charge of the Stud y 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 . Storag e The following will be stored in the archives of the Yokohama Laboratory . 1) Protoco l 2) Final report 3) Raw data 4) Test substanc e 5) Reference substance 6) Other requirements

~---- .

-6-=-

~~~

### A05P13~ Contents page Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . •- 8 ----------------------------------------••----•- ----------------------- 1 . Test Subst an ce 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 . Test Procedure 15 3 .8 . Calculation of Results 1 6 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 Concentratio n in 100% Mortality 17 4 .5 . Toxicological Symptoms 17 4 .6 . Appear ance, Temperature, Dissolved Oxygen Concentrations an d 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 Summar y Sponsor Sumitomo 3M Limite d Study Title Acute Toxicity Test of MTDID3663 with Medaka ( Oryzias latipes) Study No . A05039 7 Methods The study was conducted in accord ance 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 hour s 3) Test concentrations : con trol, solvent control, 0 .540, 0 .960, 1 .70, 3 .00, 5 .40* mg/L (nominal concentr ation) * : the maximum att ainable concentratio n Factor : 1 . 8 Solvent concen tr ation : 95 μL/L (Tetrahydrofuran) 4) Volume of test solution : 5 .0 L/vesse l 5) Number of vessels : 1 vessel/concentration 6) Biomass loading : 10 fish/concentration 7) Temperature : 24 ± 1 ° C 8) Light : fluorescent ligh t ( 16 hours light (1000 lux or less)/8 hours dark

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

LA ~3AuDg :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 Identificatio n Name of the new chemical substance (by IUPAC nomenclature ) Altern ate 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 

## S 

## ANITIZED chemical substanc e subjected to the study Name and content of impuritie s 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

--i6-- 

## LAAURA-/

1 .2 . Sourc e 

### Supplier : Sumitomo 3M Limite d 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 Specie s 1) Common name : Medak a 2) Scientific name : Oryzias latipes 3) Source : Tsunashima Fishing Co . (18-1 Tsunashima-nishi 5-chome, Kouhoku-ku, Yokohama, K anagawa) 4) Date of receipt : June 28, 2006 5) Lot No : : 06-H-062 8 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 subst an ce : 96-hour medi an 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 ± st an dard deviation=0 .86 ± 0 .27 mg/L minimum value-maximum value=0 .44-1 .5 mg/L 9) Age : Less th an a year after hatchin g 10) Acclimation period : July 19, 2006
- August 7, 200 6 Mo rtality of fish du ring 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 aquari um . 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 : fl uorescent light , 16 hours light (1000 lux or less) / 8 hours dark Feed : TetraMin, TetraWerk e Feeding amount : Approx . 2% body weight of fish/day (until 24 hours before exposure .)

_-- -- 1 2

~

### A05 8 3 3~

3 . Test Method s 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 hour s 3) Volume of test solution : 5 .0 L/vessel (Biomass loading : '_5 1 .0 g fish/L) 4) Number of vessels : 1 vessel/concentratio n 5) Biomass loading : 10 fish/concentration 6) Temperature : 24±1° C 7) Dissolved oxygen concentration : ? 60% of the saturation (not aerated) 8) pH : not adjuste d 9) Light : fluorescent light , 16 hours light (1000 lux or less)/8 hours dark 10) Feeding : non e 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-PIZ O!,

~

### A050'~

3 .3 . Apparatu s 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 R esult of range-finding test (1 vessel/concentration, 5 fish/concentration ) Concentratio n (mg/L) Mortality for 96 hours (%) Toxicological symptoms 0 .0540 0 none (7- - 0 .540 0 non e 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'\

# Aos 8R

3 .6 . Analysis of Test Solution s 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 c an not s wim ( i n cluding lying and apparent death)
.

~~ d

### AU-1017

3 .8 . Calculation of Result s 1) Determination of concentration of test subst ance for calculating median lethal concen tration (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 fr om the number of dead fish and biomass loading ( 10 fish), and median lethal concentration (LC50) was determined according to the following table . Mo rtality at maxi- > 50% <50 % mum concentration LC50 is selected among calculations

using the probit method, movin g Detennination average method and binomial method . Estimate d { method of LC50 95% confidence limits are calculated if concentration range . possible . Concentration- Included in the repo rt . Included in the repo rt . mo rtality curv e 3) Highest concentration in 0% mo rtality and lowest concentration in 100% mo rtality From the results at 24, 48, 72 an d 96 hours a ft er the sta rt of exposure, the highest concentration in 0% mortality and the lowest concentration in 100% mortality were determined if possible . The highest concentration in 0% mo rt ality is the highest concentration in which no dead fish was observed, while the lowest concentration in 100% mortality is the lowest concen tration in which all of fish were dead .

16 -

~~~

### A0A3?~

4 . Results and Discussio n 4 .1 . Circumstances That May Have Affected the Reliability of the Test Result s 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
. 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 . Highest concentration in 0% mortality after 96-hour exposure : >4 .90 mg/L ; t--- Lowest concentration in 100% mortality after 96-hour exposure : >4 .90 mg/L
.

4 .5 . Toxicological Symptom s 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 Solution s 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 .9 0

(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 Substanc e Nominal Me ana 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 mea n



### ---

--~9- ~ 

## ~y~l S

# ao M67

Table 3 Calculated LC50 Value s

Exposure LC50 95-Percent Statistical Period Confidence Limit s (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 Value s 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 .9 0 . . .. .. ... .. ... .. . . ... .. .. .. .. .. ... .. .. . .. .. ... .. .. .. ... .. .. .. ... .. .. .. . . ... .. ... .. .. . .. .. .. .. .. . . ... .. .. . . . .. .. . .. . . . .. .. ... . ... .. . .. .. .. .. ... ... .. . .. .. .. . .. .. . .. .. ... .. .. . .. .. . .. .. .. .. ... ... .. .. .. .. ... .. .. .. . .. .. .. .. .. .. .. . .. ... .. .. .. ... .. . . .. .. .. . .. ...

. ... .. .. . .. . . . .. .. ... . . .. .. .. .. ..7

... . .2 ... .. .. . . . .. .. .. . .. .. .. .. ... .. .. .. .. . . >4 .90 ~ >4 .90

. .. .. . . .. .. .. .. .. .. ... .. ... .. .. .. .. ... .. .. .. ... .. .. .. ... . .. .. ... .. ... .. .. ... .. ... .. .. . .. .. ... .. ... .. ... .. . .. .. .. ... .. .. ... .. .. .. .. . . .. . . . .. .. .. ... .. ... .. .. . . . . .. ... .. .. .. ... 96 >4 .90 >4 .9 0

--24--- ~ ~ ~p ,

### AO % 397

Table 5 Observed Toxicological Symptom s 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 .

~---

- -2 3-- L_\__17p . 31 A050397

x o U~ Ui UI Ui U' U~ U 

### ON < < V U ; U ; U~ U{ U ; U U ` • o ( !

•~ 3 0 3 ' ~ ( s

U~ Ul U

o 00 w v Q ~ o U' U~ U~ Ui U~ U : U

~ ¢ f {

C V{ U~ Vi Vi UF V~ V Ln 

# {

N ! € f ! E n o U ; Uj Uj U! UI U~ U y Uj Uj u U UI Uj U I .

" f cj ) 'i ~yQJ~
0 2 cc y b~O 

# I n o ~ j ~ . ~ : • N li j bA~o ° a ~ , ~ •~ . ~ U w tn na, ~•~~ o~ o~ ' ~ ( F, 3 0

~ .~ • ~ r- to c U{ o' o i C s c o U - ~,o o 0 0 z tn o, r,- o " t o >~ o~ O . .~ M I vl U
F~ --~3---p . 32 A050397 ! s O~ ~ L ! N N p 1 y C J C ~ O 

### ' ! O 

### , O 

### , Cl , N i M M : f'1 . d'i 'c r Nj NI ~ N~ N~ N ; N O~ Oll r

! 0 oo arn ! Ni N( N~ N N ; N N O !

•- 0 NI N~ N~ Nf N! NI N

! (

. . 3 oI p~j oj o Cii NI ~~~ N~ N~ N~ NI NI NI N a . O CO O~ O~ O~ O~ 01 o !

f''1 ml ME MI ~i -~1' F O N~ N~ N ; Ni N N ! N

N1 Na N! NI Nj Ni N a ! 0 o N -p O : Q~I O~i o! oi oi 0 W cfi ; Mi M ; ~I d- ~ t ~ N ! NI N! N~ N! N N ! ! z 0 o
~ i 3 : p ~j MI o 0 0 o ~ I ~

N NI Ni N' N~ N~ N O o
o C~V C/) o O

. o o CO b1 N V F M~ eF~ O~ CIS 'C7 O cO

d 

## ~- ~ 

## i j ~

## i O Oi N : c C1 3 O i0 . En 41 0 O Q, ,= to 0 fn
~ y E-~ cd U

c o o Cd o o~ U o~O, ~i oo, ~ Z 0 

## > ol o ; kn

0 ~ ~ ~ l! I F~ 2 4 

# LA -I c*\p . 33 

### A050397
M 0

### 0 C7\

3 M~ N! N€ N~ .--! O N 00! 0 6 00 00~ 00j CC 0 0

6 ; c pA ! ! E

N~ NI N ~ 00 : 00 00 00 ; 00 ! 00 00 0 0 U x ~ ! t I O ,

q M~ M1 M~ N~ Ni •-"! '--' ~ r 00 00 ! 00 , 00 ~ 00 0 0
-d C N ~10 116 i

O ? O-+~ M : MC MI N~ r+j ~-+! 01 i- '

00 i 00 ' 00 0 0 ! 00 061 l~ G1 . ! CL r A O { O~ 1N ~ ~ En G ~ . ~` ! ~ ! n O i i~ O M3 V1~ O C~ • U m U~ i ct~ t~f M~ ~' O~ cyd O
~ NI

b ` f •~ .~ ° ca CD CD ~ 1 ~ a3 r_ O

a cz ij ~~ rnf ° ° c, > o, oi M! t n 00 U f o

~ E-'

~~ ~

### ~0 5 34 

### 039 7

O 

### t-- r~ ! ~ 

### ! r- C-- t- : t 

### -~ E rv ~ ~ ' c X rn ( ( ` ~ Cd 

### 4 3 E 3 i tti y d ~t~ d O a x E ! c . ,

00 '~ N ~ •-• ! •--~ 1 •-- ! • ~

kn o . 'C3 N ~ ` •-- : ( •--~ ~ •-+ ,-- : ' •-• ~

v)E 

### • !
cn

° /-• ~ ~' ~' M ~F ~ ~ 'Ly N

En k o En > 

## oI ~ 3 bb " f U_ ! C13 r- o Z ol ~r~ ~o ot o ` C E U a> ~n rn t-l o "zt ; 0 > O~ o r•~ ri ~n U 0 UO ; '0 cc3 E-~

' ! .- 26 ~~ `

### A05 PjA

Figure 1 Concentration-Mo rtality Curve

100 90 80 70 ,-, 60 50 40 30 20 10 -t-- 24 hou r 0 ~ 48 hour 0 .1 1 10 x 7 2 hour

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

~---

~---

~~

### Au P u ; ~ i

Appendix 1
Infrared Absorption Spectra

~ j---,

~5-- , 

# ~~3p . 37



## ~ 

# , ~

.1~ 

# S rcx. . 

# , -~~ 

# 010 ~ Sa ✓` 

# 0- \ p 

# V-,,- e . C, vp~



## SANITIZED



# y<~ L\AUS V3 $$



## Appendix 2 Dilution Water Quality

~-36'-- 

# LA lz ;~

### AO-M67

Table A-2 Dilution Water Qualit y

Parameter Concentration BOD <2 .0 mg/L COD <3 .0 mg/L PH 8 .0 (20°C) Coliform group bacteria N .D . Oil N .D . Cadmium <0 .01 mg/L Cy an ide 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 .1 mg/L Tin <1 .0 mg/L M an ganese <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

~ . Appendix 3 Preparation of Test Solutions

~--

r3~-- 

# LA $ 

# -1tiv~ PAI

Preparation of Test Solution s 1 . Preparatio n Preparation of stock solution I Test substance __ : :> 540 mg - :

_ ------- - - -- - - - - - Solvent
--- > te trahydrofur an - - - ------------------- --- -----•----- --•------------------------------------------------------ Final volume -- - > 10 m L ---- ----- - --- --- - -- ------ - - - - - - - -- ---- Vessel ----> 10 mL volumetric bo tt l e ------- --- - - - - ---- - ---- - ------------ - - - ---------------------------- Concentration
- -- > 54000 mg/ L -- - --- - -------------------------------- - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixing method
- ---> inversio n ----- ----- - -------------------------•-------------------------------------------------------------- Preparation of stock solution I I Stock solution I _____ :> 550 μL

--------------------- ----- -- -- -- - Dissolving-agent ---- > dilution wate r - - ----- - - -----------=---------------------- - ----------------------------------------------- 2 Final voluine ----> 5500 mL x -- • in ( e Vessel
---- > 5 L media bo tt le

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

> 5 . ----------------- __ :40 mg/L -------------------------------------------•-------- -- Solvent concentration -_ __ . > __ 95 μg/ L Mixing method ---- > add•stoclk-sol . I to dilutiori wafer witli sfu-rfiig-ancf --------------

---------------- sonicate 10 min with stirrin g -----------------------------------------_ (sealing) Preparation of dissolving a g ent Solvent -- _ -_ ____ > tetrah~drofuran ----- ------------------------------------------- Solvent ( volume) --

- -

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

- -

0 ----- tt. L__________________________ •------------------- ------=----- ---

--- -- --------------------- Dissolvin~=agent _-- :

_ :_> dilution wate r

---------------------------------------------------------------------- Final volume 50 mL --- - ------------------------------------------------------------------------------------------------- Vessel
---- > volumetric bo tt l e - - - ---------------------------- ---------------------------------------------------------------------- _Solvent -----------------------~9500 0 --------- μL/L ----- - -- --- ----- - - - Mixin~ method __ :_ _> inversio n - -- - - -- -- -- --- -- - - - 2 . Preparation of test solution Stock solutions II an d 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 . agen t (mg/L) (abbreviation) (mL) (mL) (μL/L) control C ----> 0 0 0 solvent control SC
---- > 0 5 .00 9 5 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) Condition s • System Gas chromatograph/mass selective detector with headspace sampler , Agilent Technologies, Inc ., No . 1 Gas chromatograph (GC) : 6890, Agilent Technologie s Headspace sampler (HSS) 7694, Agilent Technologies Mass selective detector (MSD) : 5973N, Agilent Technologies Work station : ChemStatio n • 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 mi n Loop equilibration time : 0 .2 min Inject time : 0 .2 min Vial parameter : Shake 2 (HIGH) MS D 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 Curv e 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 mi x GC/MS analysis* 2 1 : A4 grade in JIS K055 7 *2 : Measured value represents 1/100 of concentration of standard solution
. 3 . Detection limi t 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 Solutio n (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) mi x 

### GC/MS analysi s * 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 tes t 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 Curv e

No . Concentration Peak Area

(mg/L) (count) 1 0 0 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 2530180 0 { 12 2 .00 50711191 13 5 .00 133789350 Y= 26,544,313 X 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



# ~

SANITIZE D



# y~~ - 'A 

# °

\?~

### p . 48 A05040 1



## FINAL REPORT M .S .I . Report No . A050401 Growth Inhibition Test of MTDID366 3 with Pseudokirchneriella subcapitata

Submi

### tted to : Sumitomo 3M Limited

Prepared by : Mitsubishi Chemical Safety Institute Ltd . (M .S .I .)

August 31, 200 6

---~-~

### 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 Standard s

Yokohama Laborator

y Mitsubishi Chemical Safety Institute Ltd
. Sponsor Sumitomo 3M, Ltd
. Study Title Growth Inhibition Test of MTDID3663 with Pseudokirchneriella subcapitata Study No . A05040 1 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 Laborator

y 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 Directo r 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, 200 6 ~ 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, 200 6 

### Final repo rt : August 31, 2006 August 31, 2006 Quality Assurance Personne l 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 Titl e Growth Inhibition Test of MTDID3663 with Pseudokirchneriella subcapitata (Study No . A050401 ) 2 . Purpos e The growth inhibition test of the test substance to algae was conducted in order toevaluate the toxicity of the test substance for notification under the Chemical Substances Control Law of Japan
. 3 . Test Guidelin e The test method relating to new chemical substances "Alg a g 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 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 . Sponso r

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 Laborator y Mitsubishi Chemical Safety Institute Ltd
. 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 8 . Study Directo r Tatsuhiro NIINO, Ph .D . Ecological Chemistry Division of Yokohama Laboratory 9 . Personnel in Charge of the Stud y 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 . Dat e 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 . Storag e The following will be stored in the archives of the Yokohama Laboratory
. 1) Protoco l ( 2) Final report

3) Raw dat a 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 1 1 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 1 4 

### 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

~-- = ~~s

### 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 hour s 3) Test concen tr ations, nominal : control, solvent control, 6 .30* mg/L * : limit test at the maximum a ttainable concentration Solvent concentration : 94 μL/L ( Tetrahydrofuran) 4) Volume of test solution : 100 mL/vesse l 5) Number of vessels : 6 vessels/test groups 6) Biomass loading : 5x103 cells/m L 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 subst ance in test solutions and culture s

The proport ion 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 concen tr ation reduction is volatilization from the test solutions . Inhibition concen trations were estimated based on the average of measured concentr ation (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



### --5-- So b

### A05040 p • 56

3) Inhibition concentration by comparison of areas under the growth curve s 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 . Identificatio n 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 m anufacturing ) Molecular weight ~+ N''~,~E~ Purity (%) of the ne w chemical subst an ce subjected to the study Lot number of the ne w chemical subst an ce subjected to the study Name and content of impuritie s Vapor pressure Solubility in water I-octan oUwater partition coefficient Melting point Boiling point Appearanc e at room temperature Stability Solubility i n solvents and the like

~--~~- 

## J~©~

### A0504(Pl• 58

1 .2 . Sourc e Supplier : Sumitomo 3M Limited 1 .3 . Storage and Confirmation of Stabilit y 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 Specie s 1) Organism : Unicellular green algae 2) Scientific name : Pseudokirchneriella subcapitata 3) Strain designation : ATCC2266 2 4) Source : American Type Culture Collection 5) Date of receipt : June 20, 199 6 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, 2006During the pre-culture, exponential growth of algae was observed . (The conditions were the same as the test
. )

--~1-- 

## So~

### A05040P• 59

3 . Test Method s 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 Condition s 1) Exposure procedure : static, closed system, and shake culture (100 rpm) 2) Duration : 72 hour s 3) Volume of test solution : 100 mL/vessel 4) Number of vessels : 6 vessels/test groups 5) Biomass loading : 5xl 03 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), continuou s 8) pH : not adjusted 3 .2 . Mediu m 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 . Apparatu s 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 Concentration s 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 contro l 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~ x10 0

~ 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 Solution s The method of preparation of test solutions is shown in Appendix 3 . Control groupcontained 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 Procedur e 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 andrearranged 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, besidesvisual 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 curve s 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 growt h 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 " - t i 

### 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 measuremen t tn : time ofnth measuremen t The percentage reduction in average growth rate at each test subst ance concentration ( Iμ) was calculated according to the following equation .

X100 μ c : 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 cu rves 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 curv e 

### No : nominal cell concentration ( cells/mL) at time to NI : measured cell concentration (cells/mL) at time tl N,, : measured cell concen tration (cells/mL) at time t„ tl : time of first measuremen t 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' x10 0 A, A ,, : area under the growth curve at control (or solvent control ) At : area under the growth curve at each test substance concentratio n3) 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 ~~ .

### A05040 p • 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 concentratio n Graph of concentration-growt h inhibition rates curves (plotted not included in the report on the semilogarithmic graph ) Determination method of EC50 estimated concentration rang e 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 variance s

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) .

~~~-- • 

# S~S

### A05040P• 65

4 . Results and Discussio n 4 .1 . Circumstances That May Have Affected the Reliability of the Test Result sThere 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 15 2 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 curve s 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 Solution sThe 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 Culture s

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 .000 2 . .. ... .. .. .. .. ... .. .. . .. .. .. .. .. ... .. .. _. .. .. ... .. .. .. ... ... .. ... .. .. .. . .. .. ... .. ... .. .. ... .. ... .. .. . .. .. .. .. ... . . .. .. .. ... .. .. .. ... .. .. . . ... .. .. .. . . .. .. .. ... .. . . . .. .. .. . .. . . . .. .. ... .. ... ... .. .. .. ... .. ... .. .. ... .. .. . .. .. .. .. .. ... . ... .. .. ... .. .. .. .. . . . .. ... .. ... .. ... . . .. . . ... ... .. ... ... .. . 6 .30 5 .34 0 .553 0 .433 0 .424 1 .01

(85) (9) (7) (7) (16) a : time weighted mean

S ~~

### A05040 p ' 68

Table 2 Cell Densities of Pseudokirchneriella subcapitata during the 72-Hour Exposur e 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 71570 0 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 71270 0 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 83470 0 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 mea n SD Standard deviatio n * : Nominal initial densities



## S ~"1

### A0504(9t 69

Table 3 Growth Rates of Contro l
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 .052 4 11 0 .0673 4 0 .0862 0 .0553 0 .0692 5 0 .0781 0 .0664 0 .065 7 6 Average SD CV(%)

0 .0860 0 .0595 0 .064 5 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 : Coef

fi cient of variation

~

~ 

## S ~ .C~ ,

### A05040q ' 70

Table 4 Growth Inhibition (%) of Pseudokirchneriella subcapitat a 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 1378000 0 2 0 .0688 12671000 3 0 .0689 12678000 Control 4 0 .0703 1367600 0

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 1344100 0

4 0 .0691 12930000 5 0 .0700 14096000 6 0 .070 0 13393000 -___-_______-- --- -- -------------_- ._ ._.. .__------- --------------- -Average 0

.0694 - 13429000 - SD 0 .0005 452000 1 0 .0711 14363000 6 .30* 2 0 .0698 12892000 [1 .01] 3 0 .0714 1456700 0

4 0 .0714 14190000 5 0 .0693 1303800 0 6 0 .0704 1347200 0 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 deviatio n 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 NOE C

Based on I , (0-72h) value (Growth rates ) ErC50 (0-72h) 95-Percent NOECr (0-72h) (mg/L) Confidence Limits (mg/L) (m g/L) >1 .01 -- >1 .01

B as ed on IA (0-72h) value (Areas under growth curves ) EbC50 (0-72h) 95-Percent NOECb (0-72h) (mg/L) Confidence Limits (mg/L) (m g/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 calculate d 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 { [Me an a 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 mea n Table 8 Appearance of Test Solutions after Preparation

Nominal Concentration Suspended Floating Oil y [Meana Measured Conc .] Precipitation Color solids solids Subst an ces (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 subcapitat a

(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+0 5

1 .0E+04

I .OE+03 0 24 48 7 2 Time (hr) Value in legend is given in the nominal concentration .

-26 S

### A050407• 74

Appendix 1

Infrared Absorption Spectra

{

- 2 7 S )-S ;p . 75



## SANITIZE D

~s 2k",

### A05040p• 76

Appendix 2 Composition of Medium

(

-- z'u}--

~A05040P • 77

Table A-2 Medium
Nutrient salts Concen tration (m g /L) H3BO3 0 .185 MnCI 2 • 4H 2 O 0 .415 ZnCtz 0 .003 FeC13 • 6H 2 O 0 .08 Na2EDTA • 2H2 O 0 .1 CoCI 2 • 6H2 O 0 .0015 Na 2MoO4 • 2H,O 0 .007 CuC12 • 2H2O 0 .00001 CaC1 2 • 2H2 O 1 8 NH4CI 15 K2 HPO4 1 .6 NaHCO3 50 MgCI2 • 6H2 0 12 MgSO4 7H 2 O 15 pH 8 .3

--38 ~

~~~

### A05040 p • 78

Appendix 3 Preparation of Test Solutions

5 ~°~

### A050404 79

Preparation of Test Solution s 1 . Preparatio n Preparation of stock solution I Test substance
---- > 630 mg - - - --- - ---- -- --- -- -- ---------•-•--•-- Solvent
---- > tetrahydrofur an - ----- ---- ---------------- -- ------ ------ - ----------------------------------------------- v inal olume
---- > 10 m L --- ------------------------ ------------------------------ I ---------------=-------------------------• Vessel ----> volumetric bo tt le

--------------- - - - ----- -- --- -- --------------- ---------- ----- - - - -- - ---- --- - - Concentration
---- > 63000 mg/ L . ._ . .--•-------------------------------------------------------------------------------------------------• ixing me th od -_ : _- - ----- stir by overtu rn ing with tight stopper -- -- - Preparation of stock solution I I Stock solution I ----> __ _ ____ ____ _ ---------------------•------•-- . 107------μl=-------------- Dissolvin g agent - - > medium, 23±2b- -- --------------- --- . . .-- ----- ----------------------------------•-----------• Final volume -- - > 1070 m L ---- --------------------------- -------------•---------------------------------------------------------• 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 an d sonicate for 30 mi n with ti ht sto e r -------------------•-------------- -_ - •---~------~p --------------------------------------------- . . Preparation of solvent solutio n --Solvent-------------------
-- : > - ------ - tetrahydrofur an - -----------------------•-----------

- collected volume ----> 94 μ L -- - - - -- - - - - ------------------ • ----- b__________ . ------------------------------------- ---Dissolvin~ a~ent -- -- --___> medium, 23f2 C ----- - -- - - ----- - - ------- -- - --------- Final volume ----> 1000 m L ---- -----------• ---- V essel --- - ----- -- - ----> ----- Erlenmeyer fl ask ----- - - -- - ------ . . . . - -- -- - -- ----------------------------------------------------u --------------------------------•---------------• Dissolving agent ----> 94 L/ L concentratio n ---------~ -------------------------- - ------ --- 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 an d 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 me th od ---

___>- -stir by overturning with tipht stopper ---- -- __ •---- ------- R pe licate > 6 vessels / test g roups
------------------------------------------------ - ------------------------------------------------- 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

•--3 2 

# 530

### A0504(pt 80

Appendix 4 Analysis of Test Solutions and Cultures

~

-- -

-33- 

## ~3~

### A050 4 181

1 . Gas chromatography / mass spectrometry ( GC/MS) Condition s • Syste m Gas chromatograph/mass selective detector with headspace sampler , Agilent Technologies, Inc ., No . 1 Gas chromatograph (GC) : 6890, Agilent Technologie s Headspace sampler ( HSS) 7694, Agilent Technologies Mass selective detector (MSD) : 5973N, Agilent Technologies Work station : ChemStatio n • Conditions GC Column : Rtx-200 105 mX0 .25 mm, 1 .0 μm df, RESTEK Co . Carrier gas : helium, 1 .0 mL/min (const ant fl ow) 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 mi n 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 mi n Vial parameter : Shake 2 (HIGH) MSD Temperature : Ion source : 230° C Quadrupole mass filter : 150°C SIM (Selected Ion Monitoring) : Solvent delay : 5 mi n 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 peak s

3 4 53~

### A05048182

2 . Calibration Curv e 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 limi t 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 mi x GC/MS analysi s *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 an dard solution analyzed at each an alysis . 5 . Recovery tes tBecause 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 .



## ~

--3 6 

## S 3~

### A05040T' 84

Figure A-4-1 Calibration curv e

Input Dat a No . Concentration Peak Area

(m g/L ) (count) 1 0 0 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= 26,544,313 X 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> Sp . 85

1



### C\
. . .•, c> V .,~ ,-~-

C~ .~G
. .N . s



## SANITIZED

~ ~ ~ 3 le -' ~ 3 "1

### A0583P



## FINAL REPOR T M .S .I . Report No . A05039 9 Acute Immobilisation Test of MTDID3663

with Daphnia magn a

Submitted to : Sumitomo 3M Limited

~ --

Prepared by : Mitsubishi Chemical Safety Institute Ltd . (M .S .I .)

August 31, 200 6

.-~- . 

## S'-~ ~

### A0?0399
Acute Immobilisation Test of MTDID366 3

with Daphnia magna (English version) Sponsor Sumitomo 3M Limite d Study Title Acute Immobilisation Test of MTDID3663 with Daphnia magna Study No . A05039 9 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 b y



# ---- W 1(Vt M ED

-~--- . S ; LAp . 88 

### A050399 

## Compliance with the GLP Standard s

Yokohama Laborator

y Mitsubishi Chemical Safety Institute Ltd . Sponsor Sumitomo 3M Limite d Study Title Acute Immobilisation Test of MTDID3663 with Daphnia magna Study No . A05039 9 I hereby ce rtify that this study was carried out in accord an ce with its protocol and with our standard operating procedures, an d that the repo rt accurately describes the test results . The study described in this repo rt was conducted in compli ance with the following Good Laboratory Practice ( GLP) st an dards . The st andards concerning the test facility in which the study of new chemical subst ance 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 Laborator

y Mitsubishi Chemical Safety Institute Ltd. Sponsor Sumitomo 3M Limite d Study Title Acute Immobilisation Test of MTDID3663 with Daphnia magna Study No . A05039 9 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 Directo r 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, 200 6 

### ( Observation of daphnid : 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- 

# sy3

### 99 Introductio n 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 Guideline s 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 . Sponso r Sumitomo 3M Limite d 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 Laborator y Mitsubishi Chemical Safety Institute Ltd . 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, Japan 8 . Study Directo r Midori MINO, B .Sc . Ecological Chemistry Division

--~-



# sy L~P . 91 A050399
9 . Personnel in Charge of the Stud y 

### 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 . Dat e 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 . Storag e The following will be stored in the archives of the Yokohama Laboratory . 1) Protoco l 2) Final report 3) Raw dat a 4) Test substanc e 5) Reference substance 6) Other requirements



## CSLlS

### A05 0A Content page Summary ----------------- ----------- ---------------------------------------------------------------------------------------------8 1 . Test Substance 1 0 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 1 6 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 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 p H 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

lA 

## S L\

### A05 399 Summar y Sponsor Sumitomo 3M Limite d Study Title Acute Immobilisation . Test of MTDID3663 with Daphnia magna Study No . A05039 9 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 /vesse l 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 ligh t { 16 hours light (800 lux or less) /8 hours dar k

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

SL-~c-1

### A050399

Result s 1) Median Effect Concentratio n 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 exposur e 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 exposur e 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 Substanc e 1 .1 . Identificatio n Name of the new chemical --~ substanc e (by IUPAC nomenclature) Alternate nam e 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 

# SMW E 

# D chemical subst

### anc e subjected to the study Lot number of the new chemical substanc e 

### subjected to the study Name and content of impuritie s 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 . Sourc e Supplier : Sumitomo 3M Limite d 1 .3 . Storage and Confirmation of Stabilit y 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

{- -



### - 4 3--, SSpp . 97 

### A050399 

## 2 . Test Specie s 1) Common name : Daphnia magna (Oomijinko in Japanese) 2) Scientific name : Daphnia magn a 3) Source : National Institute for Environmental Studies, Japan 4) Date of receipt : July 18, 199 5 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, 200 6 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 / wee k 

### Produced offsprings were removed daily
.

S~ ~p . 98 

### A050399 

## 3 . Test Method s 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 after 24-hour exposure ) 2) Duration : 48 hour s 3) Volume of test solution : 130 mL /vesse l 4) Number of vessels : 4 vessels /concentratio n 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 adjuste d 9) Light : fluorescent light , 16 hours light (800 lux or less) /8 hours dark 10) Feeding : non e 3 .2 . Dilution Wate r 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 . Apparatu s 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 Corporatio n 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 Concentration s 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 9 0 * : This result was judged within normal limits
.

--~4-- 

## Ss3p . 100 A050399 3 .5 . Preparation of Test Solution s 

### 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 Solution s 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 Result s 1) Determination of Concentration of Test Subst ance for Calculating Effect Concentration Concentration of test substance to calculate the effect concentrations was the average of measured concentration (time-weighted me an) . Time-weighted me an :

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 medi an effect concentration (EC50) was determined according to the following table .Immobility at maxi~ 50% < 50% mum concentration EC50 is selected among calculations Determination using the probit method, movin g average method an d binomial method . Estimate d method of EC50 95% confidence limits are calculated if concentration r ange . ~ possible .

Concentr ation- Included in the report . Included in the re o rt. immobility curve p 3) Calculation of highest concentration in 0% immobility an d lowest concentration in 100% immobility Highest concen tration 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 5p . 102 

### A050399 

## 4 . Results and Discussio n 4 .1 . Circumstances That May Have Affected the Reliability of the Test Result s 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% ImmobilityThe 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 A05039 9 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 -- Contro l 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 .85 7

(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 .5 6

(113) (96) (104) (85) (99)

a : time weighted mean New : freshly prepared test solution s 

### ~ 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 Value s

Exposure EC50 95-Percen t 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 Solution s 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 mea n New : freshly prepared test solution s Old : test solutions on 24 hours aft er 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

-~----- . ___ .----_._... .-_ ------__ __.. .^_----~----- - Solvent Control 20 .4 19 .9 20 .0 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

-- . .__. .------ .-- .-- . .. .. . 1 .80 1 .63 20 .4 19 .9 19 .9 20 . 1 3 .10 2 .90 20 .3 19 .9 19 .9 20 .1

. .-----~-- ._---_ .______---~-- ._ .__. ._------_----~---------__ .____._ .-__ .. .----__-___-_-------------__- .____----- 5 .60 5 .56 2 0 .3 1 9 .9 1 9 .9 20 .1 a : time weighted mea n New : freshly prepared test solution s 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 -__----- ._...__... .-------------------~--- _ Solvent Control ' -- 8

.8 8 .5 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 . 6 0 5 .56 8 .2 8 .3 8 .5 8 .4 a : time weighted mean New : freshly prepared test solution s Old : test solutions on 24 hours after preparation of new solutions

2 4 

# Sto3 1p .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 mea n New : freshly prepared test solution s Old : test solutions on 24 hours a 

### ft er preparation of new solutions



## -~s-~ ''~~`~

### AO ? 0399

Figure 1 Concentration-Immobility Curv e

100 90 o 24 Hours 80 - ■ -- 48 Hours 7 0 60 50 40 30 20 10 0 0 .1 1 1 0



### Concentration (mg/L)

26 S~Sp .112 

### A050399



## Appendix 1 Infrared Absorption Spectra

~



# S lplop . 113

.! _ 

## V-& V ` d 

## -, Y CG ~ I 

## o S O~/' ~ 1 C~ V\ S e- 

## C :~ V- \



## SANITIZED

S lo`tp . 114 

### A050399



## ( Appendix 2 Composition of Dilution Water

(



### 2 9 

## S lo$

### 115 A05039 9 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 .3 KCl 5 .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•2H 2 O 16 .8 ZnC12 13 .0 CoC12•6H2O 10 .0 KI 3 .25 NaZSeO3 2 .19 NH4VO3 0 .575 Na2EDTA•2H2O 250 0 ( FeSO4•7H2O 995 .5 Vitamins Concentration (μg/L) Thiamine hydrochloride 75 .0 Cyanocobalamine (B 12) 1 .0 0 Biotine 0 .750

S ~~p . 116 

### A050399



## ( Appendix 3 Preparation of Test Solutions

~--

--~1-- S

### A05 5 399

Preparation of Test Solution s 1 . Preparatio n Preparation of stock solution I Test substance ----> 560 mg - ---- - - - - - ---------- . . . > - - - Solvent ----tetrahydrofura n ------------------------------------------------•----------------------------------------------------------- Final volume ----> 10 m L --- --------•-----------•--•------------------------------------------------------------------- Vessel ----> volumetric bottle -------------------------------------------------•-----•------------------------------------------------- Concentration ----> 56000 mg/L ---------------------------------•-----•------------------------------------------------------------------ Mixing method ----> inversio n ------------------------------------------------------------------------------------------------•--•--- . ._ . . 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 bottl e ----- -----------------------------•-- - ------------------------------------------------------------- Solvent concentration ----> 9400 μL/ L ------------------------- -------------------------------------------------------------------------- MixinRmethod -_ :____ :> inversio n - ----------------------------------------------------------------------- 2 . Preparation of test solutio n 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 . agen t (mg/L) (abbreviation) (mL) (mL) (μL/L) control C ----> 0 0 0 solvent control SC ----> 0 11 .00 9 4 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 94p . 118 

### A050399



## Appendix 4 Analysis of Test Solutions

(

---3 3- ~ 

### . SZ~

### p . 119 A050399 1 . Gas chromatography / mass spectrometry (GC/MS) Condition s • Syste m Gas chromatograph/mass selective detector with headspace sampler , Agilent Technologies, Inc ., No . 1 Gas chromatograph (GC) : 6890, Agilent Technologie s Headspace sampler (HSS) 7694, Agilent Technologies Mass selective detector (MSD) : 5973N, Agilent Technologies Work station : ChemStatio n • 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) HSSTemperature : 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 Curv e 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 limi t 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 Solutio n (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 analysi s *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 A05039 9 (2) Stan dard solutions were an alyzed 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 st an dard solution analyzed at each an alysis . 5 . Recovery tes t 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 curv e

No . Concentration Peak Area

(mg/L) (count) 1 0 0 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= 26,544,313 X r= 0 .9997 1 .5E+08 ~, .
,- . ❑ 0 ~ 7 .5E+0 7 ~..
{ O .0E+00 gig , 0 1 2 3 4 5 6

Concentration (mg/L )

. ~~ ~~ (op . 123p . 124



## FINAL REPOR T M .S .I . REPORT No .A050393 Ready Biodegradability Test of MTDID3663

{

Submi tt ed 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 . : A05039 3 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 

# Uo l Date : J o Koyasu, B_Sc . Study Directo r Environmental Science Group A of Yokohama Laboratory, Mitsubishi Chemical Safety Institute Ltd .

. .~- . , S~3p . 126 

## COMPLIANCE WITH THE GLP STANDARD S Sponsor : SUMITOMO 3M, LTD
. Study Title : Ready Biodegradability Test of MTDID3663 Study No
. : A05039 3

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 Directo r Junko Koyasu, B .Sc . Sealed date : February 7, 200 6

S ~S 

# LAp . 127 

## QUALITY ASSURANCE STATEMEN T Sponsor : SUMITOMO 3M, LTD . Study Title : Ready Biodegradability Test of MTDID3663 Study No . : A05039 3 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, 200 5 Amendment 01 : November 30, 2005 November 30, 2005 Amendment 02 : January 31, 2006 January 31, 2006 In-progress study : November 17, 2005 November 17, 200 5 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 Qua lity Assurance Personnel Yumiko Kashiwagi, B .Sc . Sealed date : February 7, 2006 Hiroko Tokimi, B .Sc . Sealed date : February 7, 2006

-~-- 

## ~~'s5p . 128

1 STUDY TITLE 

### Ready Biodegradability Test of MTDID3663 (Study No . A050393) 2 PURPOS E 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 GUIDELINE S 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 N G 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 Laborator y Mitsubishi Chemical Safety Institute Ltd . (M .S .I .) 1000 Kamoshida-cho, Aoba-ku, Yokohama, Japan 8 PERSONNE L Study Director Junko Koyasu, B .Sc Environmental Science Group A Experimental Scientis t 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 DAT E 

### 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 : Februa ry 7, 200 6 10 STORAGE AND RETENTION OF THE TEST SUBSTANCE AND RECORDS The followings will be stored in the archives of M .S .I . 1) Protoco l 2) Final repo rt 3) Raw dat a 4) Test substanc e 5) Other requirements

~-- ~

-6-- S 1Z .-7p . 130 

## CONTENTS Pag e SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . .•-- . . . . . . . . . . . . . . . . . . . . . . . .----•-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- . . 8 

### 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 AFFECTE D

THE RELIABILITY OF THE TEST RESULTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 4 .2 VALIDITY
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .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 

## SUMMAR Y STUDY TITLE Ready Biodegradability Test of MTDID3663 (Study No . A050393) METHO D 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 mediu m Bottle No .2 : test substance + inoculum + mineral medium Bottle No .3 : test substance + purified wate r Bottle No .4 : reference substance + inoculum + mineral medium Measuremen t BOD based on dissolved oxygen concentration Residual test substance amoun t 

### RESULTS Meas ured 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 CONCLUSIO

N 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

## S ~°'~p . 132



## 3 TEST METHOD S 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 INOCULU M 1) Inoculum : Secondary effluent from domestic sewage treatment plant 2) Source : Naruse Clean Center, Machida cit y 3) Date of collection : November 17, 2005 3 .2 EXPOSURE CONDITION S 1)Apparatus Incubator : M-210, TAITEC (dark condition) 2)Condition s 

### 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 wate r 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 MEASUREMEN T 

### Concentration of DO, pH* an d residual test subst an ce 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 subst an ce - - - - - *At days 0 and 28, pH was measured a fter the measurement of DO . 3 .4 MEASUREMENT OF DISSOLVED OXYGEN CONCENTRATIO N Concentration of dissolved oxygen ( DO) in the BOD bo ttles 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 AMOUN T Amount of the residual test substance was measured with gas chromatograph (GC) as follows : 1)Apparatus (-- GC : 6890A , Hewlett-Packard

2)Condition s Column : RESTEK Rtx-200, 105mX0 .25 mm i .d .Xl .O V n( fi lm 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/mi n Column flow : 1 .0 mL/min ( constant flow mode) Injector : Split ( Split 5 :1 ) Detector : FID Injection volume : I μL

-~-t75 °12p . 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 MEASUREMEN T At days 0 and 28, pH was measured after measurement of DO
. 1) Apparatus : pH meter, 720A,ORION Researc h 3 .7 EQUATIONS FOR CALCULATION OF DEGRADABILIT Y 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 substanc e

(or reference substance)

-3~- 

# s°~3

### p . 135 Calculation of BO D 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/m g 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° \ 

# yp . 136



## 4 RESULT S 4 .1 CIRCUMSTANCES WHICH MIGHT HAVE AFFECTED THE RELIABILITY OF THE TEST RESULT S 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 MEASUREMEN T 

### 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 CONCLUSION S 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

z's 00 c o ~ 00 N rn o 0 0 .-, o v kn kl i ,n 16 Q a 0 0 v ° 1 :L z~ o n Q ~ O --~ M MV-~ 00 O O O 0pp o O o O'- • .--• ~[~ [~ t~ [~ V'1 tn
N
c u 00 0 0 U cC i F-1 Q Q 1--! .D . n N ~ ~ ~ R A Q Q ~



## ~ En 

## Q o En CIO Q~ O,--~ N M vi M O N O O O O M~t y -~' N ~ CV ~ N ~ O O O O O~'-''


## ~ Z 

## z O 

## z O 

## z O 

## z O 

## z O 

## z O 

## z O 

## z U

d \ ° G 00 "C S

O O N N o0 00 , .~ . . .~ N O ,• f~ N o 0 0 o M N o n pA O o O ~~~' . I M W O U C/) A ~ 0

~ 4 0 0 0 0~ cn o - U O O N M CO O O \O M O V' 1 a, O~ 'cv O O O O•--~ N y p CD o0 61 6 .\ O~, aT ~ o O o O~--En N N N N

El

Ld

~~ '+ V) v1 V7 Vl r- I~ M M . M M

~ ,!1 ,!1 \O "O 'O '~, O CA 01 O~ ~ 01 ON O U Vl .- . - . -- Ob0 O A O ~ O n O ~¢ N N N N E 4- 1 Q ~ ~ M M M M ~ ~ O M M M M ~D ~O ~ .O ~10 0 0 U\~p \D %,O \O N N -t7 U ~O ~O ~D ~1 0 trt ~ / -~ / 1 H ,i] y C M M M M M M M M

C~ Q Q CT ~ _o ~QAQn N N n Eli - f- t Cd -b 

## r~aaa E E 

## ,, p QaQQ ~ on :t : : F F• F E- ?~ E-t E- F H 

## ~ ~ ~ ~ a Cd

~ a.) ~ O tV t~i C~ i-!3 ~~ al O N N c!~ c'~ . Q ~ Z OO O O a z z z z z z z 

## zzz ~ S~4o -t-5-p . 138 U . r1 T '-r t r , '-+ t 

# , M V7 

# 00 C, O Q 0 O O t O p ♦ --4 ~1

00 ~ N 00~ t { N t / ~ t N t t 1 , i 1 'c3 Q O~

° > ~ \,o \~ O kn rn In o o pp 0 W 00 00 00 oO 00 00

b~f! ) Q L '-+ r , r p '- . i t N M kn M O N i O O O O r! " ; : t O O b D Ei Cd ~ N , (v t 1 t r t 1 N cd o ~ r e O t r t 1 t r 1 r r Cd ~ 'p
~7 o >C T:s pp N t0 \,o ~O ~O v7 ',D kn ~ N 01 Cl cd O oa 00 00 00 od 00 00 M cv t a

~ 0 b b a t4 1 4 

## °° 0

p p / N ' N 00 00 t O Oi i i O O i O O If c+)_ N t t t ~ O O O r O O C13 R~ F 4

t O r r t r r O N N cl, ~p ,fl bD t r O t t , r t t r , N Y: O A A o o b Op ~ o~n Oo~u 4 ~, pUp l0 ~O \Q " O \,R >, O C.o 00 00 00 00 00 0o t ri rl t ~ p ( l Q v' 0 El Z O ti bb ai ai Ei D U O X ~ O bb r, O O CO O ~ r e ~ OU'

O O N ' N t 1 O O t t .-- m 1 4

1 O O O O r' ^ O O ~~ c'~d . b4 b~D 0 bl) ~- j 5 ?~G k f `7 - ba O U 'CI 'CS I` c d cd c r t r t t t r t 1 1 r 11 ~~ U r~- N e N r t r r r t t r t ^, n , r t r r t r , t , t , t . t r , t ~~ o c ; O O ~ ~ '" ~ O O cl A El 00 

## o ~~ 0 0

U) N b ~ oo ar 00 oa oo 00 co cn cn ? C CA t r_n ~ O U r- V a O m CS ~ Q` ~ ~ ,` Q•. C* C d "3- °~p oa o0 00 00 oc oa 0 0 00 00 00 00 00 c~ o X o o Opp 0 0 0 0 no 00 00 00 00 0 . co co 0 0 co O O O O 0 oM U~1QLa~1 y t m Z o r C) o ti .4 z t - ~ O o o~~ a t r , ~ ~ ' a, rn o a C A N N pUq U z Z Z~ Zz Z z~ U E~+ C~ d ¢ d Q

-~ S`l1p . 139

° 0
-f1 . vUs M re c .

j V

II ~ n°

i ~ f Q C1 .

~ to 

### Raaa V 

### g~ o
~ I'D N V"1 "t m M 0 i i i i ~ N Q Q O N + ~ . bo^ o y •~ ~-+ . ~ ~ ~ Q p 

## -0 N ai A o ~ 0 0 ~ I ~ x o ~ Q o 0 0 0 0 o v~ N u U ° . . . .•-~ .-~ .-, .-, • ~ ~ Cl 

## ~
~

+~+ U ~ N QI U •y ❑ ~ U V as ~ O . cd c~ v~ U d p ~ OMi m N¢ N N d .~ t~ ~ v O 4-a cd -c-4 al 6 b U . Q U U Qy o v2 Cx a~i ~ C ° } 0 3~ a >~ ~, a U cz >, ~I ; o o $a . v ~ o «3 a`~i 9 °

v _ +~ ~ . aJ p \ ri 1--' o n +-' ~ CZ c4 ~ a~ o t ~r v, o 03 C7

~ . o o 0 0 0 ~° ~ . o TI u v ~ M M M M ~ ~ rx- + ca~ c~ cd ~ •~ ~ o~a ~ 8) n E! ~ a> ~ c ° o o - c o a~ ~ .a : I . o .~ ~ 4, ~~ 0 o --' ° • '~*~ ` o N ~ 00 0 0 x >' . a ~ ~ a i u cd V o oa c~ ~ CJ Q) U N OCt ~p M y L~+ D ~ r c d. O N c~ix a~i II -~ •~ N~ U CUi O••••~ N~ U ~,

~ ' kfj m N o ~~ ; y .•, ca :n ~ N N o Q Q oa . o0 00 00 En 0 o o . ~• ° ~ °' o° C~ a ~ . ~ ' N ~-1 U ~! N 0 .~ a)

~ ~•+
;P . t + -g VS ~ l ,~ .,_, o 0 o a . 3 j •~ ~ .a ~ ~ v> >>> u3ul-~ Ei cd 4-4 ° 0

~ ° U u ~ r : Ei

4-_ o O M ao ~ E ~ aa CJ- ° Z N C4 ti`r' o~_~ ~ ~ N N rt ri V



## z Z 

## zz - 

## ~° 40-



## ~ .S~sbp . 140

'r . ~ . N d O 

## zz 0 V 

### ~¢,aaao 0 N Cn rt M M~ v- ) C~ -Kt M o 0 N o O . - . Ci o P

- o I 0 .-+

i-~ : .Zl i-

z, i- ~ 

### U ww~x

► .C ~ .F ~"i ~ v ~"i ~ 1 * Q PC I

. + w

U bD •~ ~ . ~ ~ \O M o kn -° u) 00 O~ O ~l O~ ~ V ~~ CC N N N N +~ '+ c~~ > cn O CZj x ~, • U P . '9 X
V v ~ C :5 00 o ~ y C V ' ~ 0 o m C d o a ; M r

.r- qE l ~ ~ ~ o . cv •-• M C1 .•+ o d -t- i' a) ¢ .- co oQ 0 0 0

0 4--4 o ~ ~ ° U °

~ t4 ~ ai ai o

o 0 0 4 0 -, 4 0 - . ~ ~ o O z 6 N N t~1 tY~ ~ . *~ ~~ c~ Ej

z z z z z °

U ) > > ' Q
~ ar GCi C~ ~ F'rp . 141 Figure 1 Infrared absorption spectra of the test substance

'll



# SANITIZE D

a~ . ~©op . 142
Figure 2 Calibrati on 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 .00 0 1000 .00 0 

## ~- ~ 80a00o
~ 600 .00 0 

## ~ 400 .000
200 .000
0 .000 0 200 400 600 800 1000 1200 140 0 Concentration (mg/L)p . 143



# . ~ C ~

SANITIZED

~°°2--(,OC5p . 144

Translation Statemen t 

### Sponsor : Sumitomo 3M Limite d Title : Chromosomal Aberration Study of MTDID3663 in Cultured

Mammalian Cell s Study No . : B05134 8 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 B05134 8



## FINAL REPOR T Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian Cells (Study No . : B051348 )

July 28, 200 6 Mitsubishi Chemical Safety Institute Ltd .

~-~-- ~\~p . 146
B05134 8 

### 1 . GLP Compliance Statement Sponsor : Sumitomo 3M Limite d Title : Chromosomal Aberration Study of MTDID3663 in Cultured

Mammalian Cell s Study No . : B05134 8 This study was conducted in compliance with the following Good Laboratory Practice Standards
. ~ Standards Concerning Testing Facilities Implementing Studies Related to New Chemical

Compound s (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 Content s 

### 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
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 ( 3 .9 Study director . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 .10 Study cont ri butors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 .11 Study schedule
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 .12 Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 .13 Retention materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 

### 4 . Study Director Signature
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5 . Summary
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6 . Materi als and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 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 - 

## ~~3p .148
B05134 8 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
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 4 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
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 5 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 0 

### 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 10 .1 Unforeseeable circumstances that may have affected the quality and integrity of the data
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 10 .2 Deviation from the protocol
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 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 

### -4-- ~- \ 

# yp . 149 B05134 8 

### [short-term treatment assay (- S9 mix)] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Figure 2 Cytotoxicity in the treatment with MTDID366 3

[sho rt-term treatment assay (+S9 mix)]
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Figure 3 Cytotoxicity in the treatment with MTDID366 3

[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)

•s_,- 

## ~l'Sp . 150 B05134 8 

### 3 . Study Outline 3 .1 Titl e Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian Cells 3 .2 Study numbe r 

### B051348 3 .3 Purpos e To assess the potential of MTDID3663 to induce chromosomal aberration in a chromosomal aberration test using cultured mammalian cells . 3 .4 Guidelin e 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 GL P Standards Concerning Testing Facilities Implementing Studies Related to New Chemical Compound s (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 facilit y Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd . 14 Sunayama, Kamisu-shi, Ibaraki, Japa n 3 .9 Study director Kazushige Hori Toxicology Division II, Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd .
-G-

~~~p . 151
B05134 8 

### 3 .10 Study contributor s 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 schedul e Initiation of the study : May 10, 200 6 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, 200 6 3 .12 Retentio n 

### 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 stabl e 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) Protoco l (2) Protocol amendment (3) Test substance and control substance records (4) Cell record s (5) Study result records (6) Correspondence documents (7) Specimen s (8) Final report

-~--

~~~p . 152
B05134 8 

### 4 . Study Director Signature Sponsor : Sumitomo 3M Limite d Title : Chromosomal Aberration Study of MTDID3663 in Cultured Mammalian

Cell s Study No
. : B051348

~ Study Director :

Signed & sealed Date : July 28, 2006 Kazushige Hori Toxicology Division I) ; Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd .

k--



### -glpp . 153
B05134 8 

### 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 .

1-9-

~~p . 154 B05134 8 

### 6 . Materials and Methods 6 .1 Test substance

i 

## SANITIZED '

4 & (Z)-c>p . 155
B05134 8 

### 6 .1 .13 Stability Stabl e 6 .1 .14 Supplier Sumitomo 3M Limited 6 .1 .15 Storage conditio n 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 are a 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 substance s 6 .2 .1 Negative control substance 6 .2 .1 .1 Nam e 

### 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 _4 +



### ~~~p . 156
B05134 8 

### substance in this study
. 6 .2 .2 Positive control substances 6 .2 .2 .1 Nam e Mitomycin C (MMC) [for the test without S9 mix] Benzo [a] pyrene (BP) [for the test with S9 mix] 6 .2 .2 .2 Supplie r 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 lin e 

### CHL/IU (derived from lung of female Chinese hamster) 6 .3 .2 Supplie r Dainippon Pharmaceutical Co ., Ltd . V7 6 .3 .3 Date of receip t

May 17, 2005 6 .3 .4 Storag e 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 lin e 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

~ ~ ~~p . 157
B051348 Mycoplasma infection : Negativ e 

### 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 conditio n Culture dish : Plastic plate (diameter : 6 cm or 10 cm, Becton Dickinson and Company) Temperature : 37° C CO2 concentration : 5 % Humidity : Under moist atmospher e 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 mediu m 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 ME M (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 mediu m 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


### -1 -3 

### - 

# l0 : : 

# . .3p . 158
B05134 8 6 .5 .1 .3 Date of preparation April 21, 200 6 

### 6 .5 .1 .4 Date of receipt May 12, 200 6 6 .5 .1 .5 S9 preparatio n 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 conten t 25 .48 mg/mL (final concentration at the cell treatment : 1 .27 mg/mL) 6 .5 .1 .7 Storage conditio n -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 mi x 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 μmo l ~ KCl 33 μmol

Puri fied water remainde r 6 .6 Preparation of the test substance solutions and positive control substance solution s 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

~~~p . 159
B05134 8 

### test substance preparation from preparation to use was as follows . Cell growth inhibition test : 35 to 45 minute s Chromosomal aberration test : 20 to 55 minute s 6 .6 .2 Preparation of the positive control substance solutions 6 .6 .2 .1 Preparation of MMC solutio n (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 solutio n (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 tes t 6 .7 .1 Treatment conditio n -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 a t 

## (~2Sp . 160 B05134 8 

### 5000 μg/mL, which is required in the applied guideline, and the concentrations desc ribed in Section 6 .7 .2 were selected . 6 .7 .4 Treatmen t ( 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 Test substance 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 sho rt -term treatment assays, the surface of the cells was washed three times with MEM after 6-hour treatment and incubated in 5 mL of fr esh 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 fr om the formula of the line through the two data points showing cell growth indices higher and lower th an, 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 . -}f-

~p .161
B05134 8


### 6 .8 Chromosomal aberration test 6 .8 .1 Treatment conditio n -S9 mix assay, +S9 mix assay and 24-hour assay 6 .8 .2 Test substance concentratio n 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/m L 24-hour assay : 1250, 2500, 5000 μg/m L 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 Treatmen t 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 specimen s (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

~

~~~p . 162
B05134 8 

### 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 inde x (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 Observatio n 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 Observatio n 6 .8 .8 .2 .1 Method for coding of the specimen s 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 ,4-8- 1o2!?~p . 163
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) Fragmentatio n 6 .8 .8 .2 .5 Ga p 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 Judgmen t 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 tes t 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 thep . 164
B051348 test substance treatment group and increased dose-dependently . 

### 6 .8 .9 .5 Statistical analysi s No statistical analysis was performed . 6 .9 Expression of the result s 6 .9 .1 Tabl e (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 Figur e 

### (1) Cell growth inde x (2) Incidence of chromosomal aberrant cells

-zf - l0 3c~p . 165
B051348 7 . Result s 

### 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 Conclusio n 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 .e31p . 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 Note s 10 .1 Unforeseeable circumstances that may have affected the quality and integrity of the dat a None 10 .2 Deviation from the protocol None

-22-

~~~p . 167
B051348 Table 1 Results of the cell growth inhibition tes t 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 sulfoxid e P : Precipitation of the test substance was observed in the treatment medium at the beginning and the end of the treatment .

~- 

## tp 33p . 168
B05134 8 0 0 ~ o 0

v o° ° o C° o C° C o o C°° C o° ° C o o C o O

C W o C ~

O O O O O O O O O O o O O O O O O° O o O O O

~ 4 0 0 0 0 0 0 o c c c E a o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 z o~Soss°°ss°°SSSSSSSSSSSSS888SSSSSSSSSSSBSSSS
q C! ° o In R ~ . o r C v .9 .1 . - Ti . . . . . . . I- T TITITI

y a ° o m N c N . Ti I O N o ^

a~+ ^ ^ I o 0 0 ^ ^ ^ 0 r~ ~ o 0 0 R o 0 4 o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 G~ C ~H+H ~R E „ o Q o -~ o o „ ,- -, o d ~~y ~ o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . d

6 6 6 m U m a ~ s . b a> 0 .5 14 0 6 6 .5 6 ~ o ~' Z y ''~ eo eu E ° 5 3 ^ n o o .~ o g N °i U 60 ~ ~ p P W a .~ o r m Ea~ ° g cs ~~- o 0 0 0 0 0 a'Oi F _ 5 E

~a v 1y b U o000 0- . o .- . . ._o .- .o oo^~m ooo .,o . . ^~N .5•5 E - No v o00 -o .-ooo~c a . ra ~ S8°o8°08o°°oS°o°o°oSSSB°oo°°08°o°oSSBo°8 °°0°°0888o° °os8o°S°o8 0 w O _ -~ N _ •-• N _ •-• N . . E W ~ O z d ~, 3 a o b ^ a 30
°

z e w I I I I I I I + + } + } ,$ -T C C ~ v 5 0 0 ~o m m ro m m w m oo m m m m Q q

. ~ ~ i i i i i i i i ° 6 ^


## l~3p . 169
B05134 8



### A v

m ^~

~ a z wb

y ~ x R S d C G A A L z °

N <
y F

•~ C G •~ I
0 0 o \ m u s m ° x V U V •O p d N

7 S .IY~ c U . X 1

E" z E a

U d ❑ p m
o - < z U L b y

Q C •~
. . w F ~O o 0 00 0 °~ a ~e n~ T V o U o o z w" G

~~,~ o 0 0 0 o Q~_p , E a a 

# cQ 3 sp . 170
B05134 8 

### Figure 1 Cytotoxicity in the treatment with MTD ID 3663

[sho rt-term treatment assay (- S9 mix) ] 150 -O- cell growth inhibition test

- 0-- chromosomal aberration test 10 0

. . ., ao 50

. . . . ., U 0 0 1250 2500 3750 5000 Concentration ( μg/mL ) 

### Figure 2 Cytotogicity in the treatment with MTDID3663

[sho rt-term treatment assay (+S9 mix) ] 100

,-- . ~ 0 ~
~ a ~ . . . ~ 5 0 3 0 ~ . bn ~ -4- cell growth inhibition tes t U --*-chromosomal aberration test 0 0 1250 2500 3750 5000 Concentration (μg/mL )



### ~03~p . 171
B051348

Figure 3 Cytotoxicity in the treatment with MTDID366 3

[continuous treatment assay] 150 --o-- cell growth inhibition test .-~ -0 - chromosomal aberration test

. . . . ~ 100 a c

~ ~ o b1D 5 0 . . U 0 0 1250 2500 3750 5000 Concentration (μg/mL )



## lP 3'~p . 172
B051348 Figure 4 Incidence of cells with structural aberration s

in the treatment with MTD ID 3663 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

. . ~ W W 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 MTD ID 3663 in Cultured Mammalian

Cell s Study No . : B05134 8 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 Substance s (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 Protoco l 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 procedur e Preparation of the test substance Tomoko Kanae May 15, 2006 May 15, 2006 May 15, 2006 solutio n 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 inde x 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 K an ae Ju128, 2006 Ju128, 2006 Ju128, 2006 Quality Assurance Manager : Signed and Sealed Date : July 28 . 200 6 

### Kazuo Morotom e Quality assurance unit of Kashima Laboratory Mitsubishi Chemical Safety Institute Ltd
.

-29- k4 :1 T-1p . 174



## FINAL REPORT M .S .I . Report No . A050395 Bioconcentration Study of MTDID366 3 ~ with Carp

Submi

### tted to : Sumitomo 3M Limited

~

Prepared by : Mitsubishi Chemical Safety Institute Ltd . (M .S .I .)

August 29, 200 6



### ~~ bp . 175 

## Bioconcentration Study of MTDID366 3

with Carp (English version) 

# SMI'T UED Sponsor Sumitomo 3M Limite d Study Title Bioconcentration Study of MTDID3663 with Carp Study No . A05039 5 This report is the English version of the o ri ginal, which was w ritten in Jap anese . We, the undersigned, hereby declare that this version faithfully reflects the original repo rt to the best of our knowledge . 

# ~_- ~ ~, .- • I

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

~ Approved by
Date : 

# °p

~ ---" ; -------•-----•----------•------------------------

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

### Yasuo~ Study Directo r Ecological Chemistry Division of Yokohama Laborato ry, Mitsubishi Chemical Safety Institute Ltd .p . 176 

## Compliance with the GLP Standard s Yokohama Laboratory , Mitsubishi Chemical Safety Institute Ltd
. Sponsor Sumitomo 3M Limite d 

### Study Title Bioconcentration Study of MTDID3663 with Carp Study No . A05039 5 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 Directo r



### Ecological Chemistry Division of Yokohama Laboratory

--~-- (o ~ ~p . 177 

## Quality Assurance Statemen t Yokohama Laboratory , Mitsubishi Chemical Safety Institute Ltd . Sponsor Sumitomo 3M Limite d 

### Study Title Bioconcentration Study of MTDID3663 with Carp Study No . A05039 5 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, 200 6 ( 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, 200 6 Final repo rt : August 29, 2006 August 29, 2006 Quality Assurance Personne l 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 3p . 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 Guidelin e The study was conducted in accordance with the Test Method Relating to New ~ Chemical Substances <Bioconcentration test of chemical subst ances 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 Comp li anc e 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 . Sponso r

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, Japa n 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 Directo r 

### Yasuo SAT O Ecological Chemistry Division of Yokohama Laboratory
9 . Experimental Scientis t 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 . Dat e 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, 200 6 11 . Storage and Retention of Test Substance and Record s The following will be stored in the archives of the Yokohama Laboratory
. 1) Protoco l 2) Final report 3) Raw dat a 4) Test substanc e 5) Other requirements

-6- lo LA Sp . 180 

## Contents page Summary ----------------------------------------------- 9 1 . Test Substance ------------------------- 13

---------------------------------------- 1 .1 . Identification 13 1 .2 . Source 14 1 .3 . Confirmation of Stability 1 4 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 1 6 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 2 1 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 3 0 Tables & Figures 31-72 Appendix 1 Information on the test substanc e Appendix 2 Dilution water qualit y 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 ~-lp . 182 

## Summary Sponsor : Sumitomo 3M Limite d Study title : Bioconcentration Study of MTDID3663 with Carp Study number : A05039 5 Study period : March 24, 2006 to August 29, 200 6 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 wate r 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 day s Depuration period : 8 day s 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

lQyp . 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 leve l Mean conc . in test water (mg/L) 0 .0479 0 .0485 0 .0492 0 .0497 0 .0493 BCF, No . 1 1250 982 1230 1050 1660 BCFss 1210 No .2 1360 1230 1000 1130 1100 Low concentration leve l Mean conc . in test water (mg/L) 0 .00480 0 .00486 0 .00488 0 .00492 0 .00498 BCF, No . 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 Mean conc . in test water (mg/L) 0 .0475 0 .0482 0 .0490 0 .0495 0 .0492 BCF, No . 1 1310 1030 1290 1110 1760 B CFss 1270 No .2 1430 1280 1050 1200 1160 Low concentration leve l Mean conc . in test water (mg/L) 0 .00479 0 .00485 0 .00489 0 .00492 0 .00496 BCF, No . 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 leve l Mean cone . in test water (mg/L) 0 .00491 0 .00494 0 .00498 0 .00500 0 .00507 BCF, No . 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 leve l Mean conc . in test water (mg/L) 0 .0498 0 .0500 0 .0507 0 .0509 0 .0500 BCF, No . 1 874 667 843 731 1180 BCFss 863 No . 2 970 820 673 795 796 Low concentration leve l Mean conc . in test water (mg/L) 0 .00495 0 .00499 0 .00499 0 .00502 0 .00504 BCF, No . 1 987 861 820 824 1070 BCFss 898 No . 2 860 1070 811 849 1100p . 184

Discussio n 

### 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 tissu e 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 106 0 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 tes t 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
.

~



## ~1Q51p . 186 1 . Test Substanc e 1 .1 . Identification Name of the new chemical substanc e (by IUPAC nomenclature) Alte rnate nam e CAS RN Structural formula or rational formul a (If both of them are unknown, outline of preparation or manufacturing ) Molecular weight 

## SANITIZED Purity (%) of the ne w chemical substance subjected to the study Lot number of the ne w chemical substance subjected to the study Name and content of impuritie s 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 lik e 

### All data were provided by the sponsor

--~ - ~S~p . 187



## E33 

## l .lT'1 

## MA2

.p . 188

1 .2 . Sourc e 

### Supplier : Sumitomo 3M Limited 1 .3 . Confirmation of Stabilit y 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 Tes t 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 factor s ~ (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 3p . 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 Tes t 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-082 6 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 c m Body weight : Approximately 0 .2 g 2) Acclimation Aquarium number : D-11 Water temperature : 24 ± 1 °C Period : - >_ 2 week s 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 : 5 L Water temperature : 24± 1 °C Renewal of test water : Every 24 hour s Aeration : None (Covered with Teflon sheet) Dissolved oxygen conc
. : 60% of the saturation (? 5 mg/L at 24 °C) Feeding : Non e Duration : 96 hours 4 .3 . Preparation of Test Water 1) For exposure grou

p 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 contro l 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
. 

## ~'IeSSp . 191

5 . Analytical Method s 

### 5 .1 . Apparatus and Reagents Apparatu s 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 Reagent s 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 689 0 Head Space Sampler (HSS) : Agilent 7694 Mass Selective Detector (MSD) : Agilent 5973N Work station : MSD Chemistation Condition s rGC l 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 ) FHSS 1 Temperature : Oven 60°C, Loop 120°C, Transfer Line 200°C Event time : GC Cycle Time 40mi n

Vial Equilibration Time 20min Pressurization Time 0 .2min Loop Fill Time 0 .03min Loop Equilibration Time 0 .2min Inject Time 0 .2mi n Vial parameter : Shake HIG H [MSD ] ~ Temperature : Ion source 230°C, Quadrupole mass filter 150° C SIM (Selected Ion Monitoring) method :

Solvent Delay 8 .0mi n 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 -1p . 193

5 .3 . Calibration Curv e 

### 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 methodwas 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 leve l 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 leve l HS vial containing test water IOmL is capped immediately after adding ( acetonitrile 0 .1mL . I Performed GC/MS analysi s 

### * :Purified water : Grade A4 in Japanese Industrial Standards (JIS) K0557

~- 1~5bp . 194

5 .5 . Analysis of Test Substance in Test Fis h 

### 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 scissor s Homogenized for 5 minutes at approx . 8000 rp m Filtered with suction through filter paper (S-60) on Buchner funnel A cetonitrile layer Residu e 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 m L I After added 0 1 mL portion (Dilution ratio 1000), HS vial containing purified water* l OmL is capped immediatel y Performed GC/MS analysi s * :Purified water : Grade A4 in JIS K0557p . 195

5 .6 . Blank and Recovery Test s 

### 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 tes t 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 tes t 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 tes t 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 tes t 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 .0 5 ~ 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 C5p . 196

6 . Bioconcentration Test Method s 

### 6 .1 . Test Fis h 1) Species : Carp (Cyprinus carpio) Source : Niikura Fish Far m 1217, Shimoya, Isehara-city, Kanagawa, Japan Date of purchase : July 4, 200 5 Lot number : 05-K-070 4 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 c m Body weight : Approximately 5 g Age : Less than a year after hatching 2) Acclimatio n Aquarium number : A-1 Water temperature : 24 ± 2 °C Period : > 2 weeks Food : Babygold® from Kyori n Feeding : The food was given in an amount of 2% of the fish weight
. Mortality during the 1 week before testing : <5 %

i Ip . 197

6 .2 . Test Condition s 

### 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/da y 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 : Non e 8) pH : 6 .0-8 . 5 9) Lighting : Approximately 16 hr/da y (Hf fluorescent lamp with wavelengths of 400-700 nm) 10) Exposure period : 60 day s 11) Depuration period : 8 days (By supplying dilution water without test substance) 6 .3 . Test Apparatu s 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 leve l 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 leve l Feed solution 1, 50 mL E--2-methoxyethanol Diluted to 500 mL (Feed solution 2 ) Concentration of test substance : 200 mg/L Contro l 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 3p . 199

6 .5 . Introduction of Fish, Observations and Environmental Contro l 

### 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 fis h 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 temperatur e 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 Corporatio n

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 Substanc e 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 Facto r 1) Calculation of bioconcentration facto r The bioconcentration factor (BCF) is calculated by the following equation :

BCFn = Cfn / Cw n 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 facto r 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 facto r 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-lif e 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~e5p . 201
t : Time in depuration period (day ) 

### On each of the two concentration levels, the depuration rate const an t, 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 ~Pp . 202

7 . Results and Discussio n 

### 7.1 . Circumstances That May Have Affected the Re liabi lity 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 an d 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 perio d (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 Fis h 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 lastp . 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 tissu e Peak A BCF ~_ . Part High concentration Low concentration level (day 60) level (d ay 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 lep . 204
P e 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 106 0 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-lif

e 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]

Clp . 205

Table 1 Blank test of the test substance in water A B C peak sample peak area conc . No . No . count in wate r 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