# STATEMENT

Study title:
Partition Coefficient (I-0ctanoVWater) Study of

I, the undersigned, hereby declare that this report provides a correct English translation ofthe Final Report

August 3, 2004



### Study Director:



# 000597

# CERTIFICATION

Title ofTest: Partition Coefficient (l-OctanollWater) Study of

Identification code.:

The above test was conducted in accordance with the Good Laboratory Practice Standards for industrial chemicals, "Basic standards to 

### be observed by testing facilities in conducting tests stipulated in article 4 of the Order Prescribing Those Items of the Test Relating the New Chemical Substance and Study on Harmful Effects of Designated Chemical Substances" (March 31, 1984, Revised March 1, 2000, Kanpogyo No.39. Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229. Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyoku No.85. Basic Industries Bureau, Ministry ofInternational Trade and Industry, Japan)
.
April 4. 2001
StudyDirector:

__(,,:;S;..:Jign=e=d)<.-

__



### 1 

# 000598

# QUALITYASSURANCE STATEMENT
Type ofTest Partition Coefficient (l-OctanollWater) Study Name oftest substance

This test was conducted in accordance with "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 ofthe Order Prescribing Those Items of the Test Relating the New Chemical Substance and Study onHannful Effects ofDesignated Chemical Substances", We acknowledge that the Final Report describes the methods and procedures used in the test precisely and that the results reported hereinreflect the raw data accurately, Aud' 1t and' mspection were con ducted as fillows: 0 

### Audit orInspection Items Date ofAudit or Inspection Date ofreport Protocol March 2, 200I March 2, 2001 Conduct oftest March 9,2001 March 9, 2001 Draft Final Report April 3, 2001 April 3, 2001 Draft Final Report Amendment April 3, 2001 April 3, 2001 Verification ofDraftFinal Report Amendment April 4, 2001 April 4, 2001

Date ofissue thereof April 4, 2001 Quality assurance personnel



## - _(Signed)



### - 

## I

2 

# 000599

### Title ofthe test: Partition Coefficient (I-OctanollW ater) Studyof

Test Client:

Name: Address: Test Facilities: Name:
Address: Testing Personnel:

Management: StudyDirector: Test Personnel:
Test Period: Date oftest initiation: Date ofexperiment initiation: Date ofexperiment termination: Dateoftest termination: March 2, 2001 March 

### 9, 2001 March 9,2001 April 4, 2001 Final 

### Report: Preparation: April 4, 2001 Study Director: APPROVAL OF FINAL REPORf

Study Director: August 2, 1999 (Signed)



### 3 

# 000600

### SUMMARY

# 27~ I. 

### Description ofTest: Partition Coefficient (l-OctnnollWater) Study 2. Test Substance: 3. TestMethod and Conditions 3-1. TestMethod The test was conducted in accordance with the "Commentary for the Measuring Method ofthe Partition Coefficient (l-OctanollWater) of Chemical Substances indicated" in the Order Prescribing the Interpretation and Application of the Law Concerning the Examination and Regulation of Manufacture, etc. of Chemical Substances (24th March. 1987, Pharmaceutical Affairs Bureau No. 291, and 62 kikyoku No.I71, Basic Industries Bureau (,87» and "Partition Coefficient (n-OctanollWater): 107, Shake FI

### askMethod " in the OECD Guidelines forTesting of chemicals (July 27,1995).
3-2. Testing Conditions Temperature: 23±

## I"C
4. Test Results

The log Pow value (fotal average) :2.18 The standard deviation :0.109 The difference between the maximum and the minimum value was 0.327, which was not significant. There was noenvironmental factor orabnonnal condition that was presumed to affect the reliability oftestresults. Therefore, the test results arejudgedto be fully reliable.

4 

# 00060.1

### Contents 

# '-77 t. Purposc ofthe Tcst
.............................................................................................................................. 6 

### 2. TestMethod
......................................................................................................................................... 6 3. TestPeriod
........................................................................................................................................... 6 4. TestSubstance
..................................................................................................................................... 6 

### S. Conrumation ofStability underthe Storage Conditions
..................................................................... 6 6. Measurement ofthe Partition Coefficient (l-OctanoVWater)
............................................................. 7 7. Discussion and Conclusion
................................................................................................................ 11 8. Environmental Factors Presumed to Mfectthe Reliability ofTest Results
...................................... 11 9. Storage ofTsetSubstance and Raw Data etc
.................................................................................... 12 10. Compliance with GLP
..................................................................................................................... 12 11. List ofAttached Figures
.................................................................................................................. 12 12. List ofAttached Tables
.................................................................................................................... 12



### 5 

# 000602

### 1. 

### Purpose oftheTest The purpose of this test is to conduct partition Coefficient (l-OctanoVWater) Study of to obtain infonnation on its degree ofbioaccumulation as part ofthe program to verify the satety ofa new chemical substance. 

### 2. TestMethod The test was conducted in accordance with the "Conunentary for the Measuring Method of the Partition Coefficient (l-OctanoVWater) of Chemical Substances" indicated in the Order Prescribing the Interpretation and Application ofthe Law Concerning the Examination and Regulation ofManufacture. etc. of Chemical Substances (24th March. 1987, Pharmaceutical 

### Affairs Bureau No. 291, , and 62 kikyolru No.171, Basic IndustrIes Bureau ('87», and "Partition Coefficient (n-OctanoVWater): 107, Shake FlaskMethod" in the OECD Guidelines for Testing ofchemicals (July 27,1995).
3. TestPeriod Date oftest initiation: March 2, 2001 Date ofexperiment initiation: March 9, 2001 Date ofexperiment termination: March 9, 200I 

### Date oftest termination: April 4, 2001
4. Test Substance* Name: Abbreviation: Identification code: Structural fonnula: Lot No.: Purity: 100% Molecularformula: Molecular weight: 200.05 CAS 

### No.: Properties: Appearance: liquid Storage Stored at room temperature in a darkplace conditions: *) 

### Information provided by the testclient (except for Identification code.)
5. Confirmation ofStability underthe Storage Conditions Infrared absorption spectra ofthe test substance were obtained at the beginning and at the end ofthe test, and the two spectra were found to show no difference. Based on this result. it wasjudged that the test substance remained stable throughout the test period. (Fig. 1)



### 6 

# 000603

### 6. Measurement ofthe Partition Coefficient (l-OctanoVWater) 6-1. MajorEquipment and Reagents Used 

### in the Test 6-1-1. Reagents Pure water: Prepared by means of pure water production equipment MiIIiQ-Labo and then distilled. Less than 1 Jl S/cm 

### of electric conductivity and more than 60 minutes of K2Mn04 color retention time were confmned prior to the use. 1-0ctanol :( Partition Coefficient Grade) Mitsubishlkasei Methanol (lIS Guaranteed Grade): Iuosei Chemical Isopropyl alcohol (JIS Guaranteed Grade); Kishida Chemical 6-1-2. MajorEquipment Gas chromatograph HP-5890 Hewlet-Packard Mass SpectrometerIMS-AX505H IEOLLTD. Shaker Y AYOI YGG YAYOICO., LTD. Karl FischerMoisture TitratorMKS-2IO Kyoto Electronics Manufacturing Co., Ltd. pH meterlIM-50V Toa Denpa Kogyo Co., Ltd. Electric conductivity meter CDM-83 RADIOMETER High Speed Refrigerated Centrifuges SCR-20BB Hitachi, Ltd. Water PurifierMi1IiQ-Labo Nihon Millipore Ltd. Water Stills WB-21 Yamato Scientific Inc. Chemical balance BP-210-D Sartorius Co., Ltd. 6-2. Mutual Saturationofthe Solvents Water and 1-0ctanol were mixed at the rate of 2:1, and the mixture was stirred using a magnetic stirrer for 24 hours. Next. all the quantity was shifted to a separatory funnel, allowed to settle, and separated. Water in l..Qctanol when it was mutually saturated with the water was analyzed using Karl Fischer Moisture Titrator. And, 1-0ctanol 

### in Water when it was mutually saturated with the l-Octanol was analyzed using Gas chromatograph. Then, mutual saturation was confmned. (l-Octanol concentration 

### in the water was 411mgIL, and it was beyond 400mglL. And. Water concentration in the l-Octanol was 4.94%. and it was beyond 4%.) 6-3. Preparation ofStockSolution lOOmg of the test substance were weighed by use ofChemical balance into a IOO-mJ volumetric flask, dissolved 

### in water-saturated l-octanol and diluted to the mark. (Test substance concentration: 1000 mgIL) 6-4. Determination Conditions and Method GC-MS Conditions Gas chromatograph: HP-5890 Mass Spectrometer: JMS-AXSOSH Column: CP-PoraPLOT Q 0.53m

### q, x 25mm (Film thickness 20 Jl

### m) Carrier gas: He 7 psi SPLIT flow: 50mllmin Column oven Temp.: 

### 60"C-(lO"C/min)-185"C Inj. Temp. , SeparatorTemp: 200"C Inj. Volume: 1Jl 

### L Measurement mode: SIM method (monitorion: mle83(test substance), mle45i(internal standard» Dataprocessor: DA-5000 Determination method: Absolute calibration curve method, y 

### = ax (3-point method), Decision 

### 7 

# 000604

### was made to carry out the quantitative determination of the test substance in the tested liquids by the one-point method, since the calibration curve was a straight line passing the origin. (see 6-S-3.) 6-S. 

### Measurement ofLimit Value for TestWater Analysis, Calculation ofDetection Limit Concentration, and Preparation ofthe Calibration Curve 6-S-I. Preparation ofStandard Solution for Preparation ofthe Calibration Curve A test substance solution with a concentration of 100 mglL was prepared by weighing 10 mg of the test substance by use of Chemical balance into a lOO-mL volumetric flask, dissolving it in methanol, and diluting to the mark. Then, 1 mL of the solution was transferred to a 20-mL volumetric flask by use ofa volumetric pipet and diluted to the mark with distilled methanoL Thus a test substance solution with a concentration of5 mglL was prepared. And, an internal standard substance solution with a concentration of 1000 mglL was prepared by weighing 10 mg ofisopropyl alcohol into a 1O-mL volumetric flask, dissolving it in methanol, and diluting to the mark. Then, about 80% volume of 5-mglL test substance solution was transferred to a 10-mL volumetric flask, added 10 f.L 

### L of the lOOO-mgIL internal standard substance solution by use of a micro syringe, dissolved in S-mgIL test substance solution and diluted to the mark. Thus, a solution of

### S mgIL test substance containing 1 mglL internal standard substance was prepared. Likewise, S 

### mL and 1 mL of the S-mgIL test substance solution was transferred to 1O-mL volumetric flasks by use ofa volumetric pipette and diluted to the mark with distilled methanol and 10 f.L 

### L ofthe lOOO-mgIL internal standard substance solution. Thus, solutions of 

### 2.S or O
..S mgIL test substance containing 1 mgIL internal standard substance were prepared. These three solutions containing internal standard were used as standards for the preparation of the calibration curve. 

### 6-S-2. Calculation ofDetermination Limit Value and Detection Limit Concentration The 5-mgIL test substance solution described in 6-5-1 was diluted with methanol 100 times, and internal standard substance solution was added so that internal standard substance concentration become ImgIL, and thus test substance solution with a concentration of O.lmgIL was prepared. This solution was analyzed using the determination conditions and method described in 6-4., and the measurement limit value and the detection limit concentration were calculated. The minimum detection limit concentration was 0.00536mgIL. And, the measurement limit concentration of analysis samples were calculated. Stock solution: 1.34 mg/L Partition eqUilibrium solution I-Octanol phase condition-I, 2, 3 : 1.34 mgIL water phase condition-I, 2, 3:0.0179 mgIL 6-5-3. Preparation ofCalibration Curve Analysis was conductedusing the determination conditions and method described in 6-4. The linearregression equation y

# =ax 

### + b was found by the least square method, and the regression coefficient (a), intercept (b) and correlation efficient (r) were verified. y: peak area a: regression coefficient x: concentration b: intercept r: correlationcoefficient As a result, b was not acknowledged to be significant. Consequently, calculation was made again using the regression equation y 

# =ax, and the regression equation as shown below was obtained. From that point onwards, the one-point calibration curve method was adopted for determining the recovery and the test substance in the tested liquid. The calibration curve thus obtained is shown below. y= 1.6119x(r=0.99860)
8 

# 000605

### Further, the calibrationcurve was also utilized for the analysis. (Fig. 2-1 Cnlihrntion Curve) 

### (Fig. 2·2 Calibration Curve Chromatogram) 6-5-4. Analysis ofTest Substance StockSolution 1 ml of the stock solution with a concentration of 1000 mglL described 

### in 6-3. above were transferred to a 25-ml volumetric flask by use of a volumetric pipette and diluted to the mark with methanol.

Then, 

### 1 mL ofthis solution was transferred to a lO-mL volumetric flask by use ofa volumetric pipette, and after 10 J1. 

### Lof lOOO-mgIL internal standard substance solution was added, diluted to the mark with distilled methanol. Then analysis was conducted twice under the condition of 6-4. using this standard solution. Then. concentration ofthe test substance was determined by the one-point method.

The results were as follows. Average test substance concentration in stock solution was 685 mgIL, and the amount ofthe test substance in the partition equilibrium solution was 3.43mg. (Fig.3 Chromatogram forAnalysis ofStock Solution, Table 

### 1Data table ofStock Solution) (F 

## x H I G) 

## x B

## x C

## x D

## x 100

Concentration ofstock solution(mglL)

- .-------------­ AXE/1oo0 A: Volume oftest solution (mL) B: Volume oftest solution (mL) C: Conversion coefficient* D: Transferratio* 

### E: Recovery 

### (%) F: Standard concentration (mgIL) G: Standard peak arearatio H:Test substancepeak arearatio *)The term "conversion coefficient" means a coefficient that is used in quantitative determination of the test substance 

### in terms of the non-test-substance components by subjecting the test substance to chemical decomposition. turning it into derivatives. etc. In 

### this test, the conversion coefficient was set at 1. And. the recovery rate was made 100% because analysis operation such as extraction wasn't done. 6-6. The Partition Equilibrium Test

6-6-1. Testing condition 1) Temperature: 23 

### + 1

## "C 2) The number ofrepetition: 2 times permeasurement condition 3) Added amount oftest substance: 3.43mg 4) Volume oftest substance solution and solvent, and volume ratio ofsolvents condition-l condition -2 condition -3 Volume oftest substance solution 5 5 5 Volume ofwater-saturated l-octanol 0 5 15 Volume of l-octanol-saturated water 35 30 20 (mL)



### 9 

# 000606

### 6-6-2. Partition Equilibrium Flow 1) 

### 5 m1 aliquots of the stock solution with a concentration of 1000 mglL described in 6-3. above were transferred to 6 test vessels by use of a volumetric pipette. Furthermore, water-saturated I-octanol and l-octanol-saturated water were added in the exact volume set up in the above-mentioned examination conditions to the test vessels using volumetric pipettes. 2) The test vessels were set to the shaker and shaken for 5 minutes by 20rpm. 3) The test vessels were removed from the shaker and set in the rotor of the high speed refrigerated centrifuges maintained at examination temperature. The test vessels were centrifuged by 5000rpm for 20 minutes. 4) Two phases were confinned to be separate completely. A capillary pipette was inserted in the central part of l-octanolphase in each test vessel to extract l-octanol, and less than halfamount of the phase was moved to a sample bottle. The remainder was carefully removed and discarded. 5) A capillary pipette was inserted in the lower part of the water phase in each vessel, and about 10 mL ofwater phase was extracted. Then, after wiping offthe outside ofthe capillary pipette with Kimwipe paper wipes, it was moved to a sample bottle. And, pH of remained water phase was measured. 6) According to the analysis method of6-6-3., the test substance concentrations in I-octanol phase and water phase were measured. 6-6-3. Analysis ofTest Substance in Partition Equilibrium Solutions 1) 

### Analysis oftest substance in I-octanol phase I-mL aliquots of l-octanol phase obtained by 6-6-2. were transferred to 25-mL volumetric flasks by use ofa volumetric pipette, and after 10/.t 

### Lof 1000-mglL internal standard substance solution was added, diluted to the mark with distilled methanol. Furthennore, I mL 

### of each solution was transferred to a lO-mL volumetric flask by use ofa volumetric pipette and diluted to the mark with distilled methanol.

Next, they were analyzed under the condition of 6-4. repeatedly using the 2.5mgIL standard solution. Then, concentration ofthe test substance in the I-octanol phase was determined by the one-point method. (Figs.4-1--4-3 Chromatogram forAnalysis oftest sample, Table 2 data table of I-octanol phase) 2) Analysis oftest substance in waterphase 3-mL aliquots of water phase obtained by 6-6-2. were transferred to lO-mL volumetric flasks by use of a volumetric pipette, and after 10/.t 

### L of IOOO-mgIL internal standard substance solution was added, diluted to the mark with distilled methanol.

Next, they were analyzed under the condition of 6-4. repeatedly using the 2.5mgIL standard solution. Then, concentration ofthe test substance in the I-octanol phase was determined by the one-point method. (Figs.4-1--4-3 Chromatogram forAnalysis oftest sample,

- Table 3 data table ofwaterphase) Concentration of Partition eqUilibrium 

# = (F X H / G) X B X C X D X 100 

### solution(mgIL) AXE/lOOO 

### A: Volume oftest solution (mL) B: Volume oftest solution (mL) C: Conversion coefficient* 0: 

### Transfer ratio* E: Recovery (%) 

### F: Standard concentration (mgIL) G: Standardpeak arearatio H:Test substance peak area ratio *)The tenn "conversion coefficient" means a coefficient that is used in quantitative determination of the test substance in tenns of the non-test-substance components by subjecting the test substance to chemical decomposition, turning it into derivatives, etc. In this test, the conversion coefficient was set at 1. 

### And, the recovery rate was made 100%

10 

# 000607

### because analysis operation such as -extraction wasn't done. 3) The calculation method ofPartition Coefficient According to the following formulas, the partition coefficient oftest substance was calculated from the test substance concentration in I-octanol phase and water phase. LogPow means the logarithm to base 10. Co Partition Coefficient (Pow)=-C-wCo: Concentration oftest substance in l-octanol phase (mgIL) Cw :Concentration oftest substance in waterphase(mgIL) 6-7. Analytical Results The test substance concentrations'in each phase after Partition equilibrium analyzed according to 6-6-3. are shown in the following table. pH ofthe waterphase are also shown. concentration of l-octanol phase concentration of waterphase pH ofwaterphase condi condit condit tion-I ion-2 ion-3 1 2 1 

### 2 1 2 534 mg 625 387 302 227 203 lL 4.76mgIL 3.90 2.75 2.17 1.56 0.853 6.97 7.09 7.15 7.16 7.84 7.55 7. Discussion and Conclusion 7-I.Resultofthe Partition Coefficient From the test substance concentration in each phase after partition equilibrium acquired by 6-7., the Partition Coefficient was calculated and the Partition Coefficient result table was created. The result is shown below. (Table 4 Data table ofPartition Coefficient) log Pow condition -1 : 2.05, condition -2 : 2.15. condition -3 : 2.16. 2.20 2.14

2.38 log Pow Total average: 2.18 Standard deviation: 0 LogPow[max]-logPow .109 [min] :0.327 7-2. Discussion The LogPow value (total average) oftest substance was 2.18 as a result ofthe study. The standard deviation"was 0.109 and the difference between the maximum and the minimum value was 0.327, which was not significant. Moreover, no functional group in the test substance is known as dissociable. And, pH ofwater phase afterpartition equilibrium was neutral. Therefore, these test results arejudgedto be fully reliable. 8. Environmental Factors Presumed toAffect the Reliability ofTest Results The temperature change during the testexamination period was less than 

## ± 1 degree C. There was no environmental factor or abnormal condition that was presumed to affect the reliability oftestresults.

11 

# 000608

### 9. Storage ofTest Substance and Raw Dataetc. 9-1. Test Substance The test substance for safekeeping was put in a storage container which was closed with a ground stopper. It 

### is to be 

### kept in storage in the storage room of this center for the period as stipulated in Article 32 ofthe Standards Concerning the Test Facilities As Set Forth in Article 4 ofthe Ordinance Stipulating the Items ofTest Relating to New Chemical Substances (hereinafter referred to as "Test Facilities Standards"). 9-2. Raw Data, Documents, Etc. Analytical data obtained from the test, results of measurement and observation raw data used for preparation of the final report, protocol, etc. are to be 

### stored for the period provided by Test Facilities Standards in a storage room ofthe test substance in this center.

l.Test Plan 2.Final Report 3.Records relating to the raw data, facilities, and performance oftest that are used in the

preparation ofthe fmal report (original orcopy): -Records onprofessional career and training for individual employees -Records relating to administration -Records relating to the test substance

-Records relating to guaranty for reliability -Records relating to performance oftest -Raw data -Preliminary study memos, data, etc. 4. Samples Samples for final analysis 5. 

### Other data deemed as necessary 

### 10. Compliance with GLP This test was conducted in accordance with the 

### GLP standards (March 31, 1984, Revised March 1, 2000, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs 

### Bureau. Ministry of Health and Welfare. and 59 Kikyoku No.S5, Basic Industries Bureau, Ministry of International Trade and Industry, Japan) • and the statement of the StudyDirector and the written guaranty prepared by the person responsible for reliability guaranty are attached to the fmal report.
11. List ofAttachedFigures Fig. 1 IR Spectra Fig. 2-1 Calibration Curve Fig. 2-2 Calibration Curve Chromatogram Fig. 3 ChromatogramforAnalysis ofStock Solution Figs.**4~.1** 

### and4-3 Chromatogramfor Analysis oftest sample 12. List ofAttachedTables Table 1 Datatable ofstock solution Table 2 Datatable ofI-octanol phase Table 3 Data table ofwaterphase Table 4 Data table ofPartition Coefficient



### 12 

# 000609

### 70 

### Fig. 1 

## IR Spectra
Identification code
. Sample comment: initiation ofthe test Date: 200113/5 

### Method: Liquid film Instrument: FfIIR-7000 Operator: Hirokazu Yamane**w,---------------------------------------------------~** A 80 

## f\

# ----rrv.~~\ 

# . 1\ 1

# I \

60 

# I
96T I 

### 40

# l.~ f
v·V 

### 20

### 1~0 4000 2000 Wavenl..fllbericm-l] 1000 400



### Identification code: Method: Liquid film Sample comment: termination ofthe test Date: 2001/3/23 Instrument: FT/IR-7000 Operator: Hirokazu Yamane



### 96T 80

4000 

### \ /l 

## 1

1000 400 

### 2000 Wavenumbericm-l]



# 000610

### Test date : 2001/3/9 

# Fig. 2-1 

# Calibration Curve Identification code. Type of test: Partition Coefficient Test item: Analysis of Stock Solution

Operator: Hirokazu Ymane 

## 10.00

8.00 0 '.j:i 

### ca 

## 6.00 Concentartion(mE/l) Area ratio 0.540 0.9238 2. 70 4.0881



# .

- 

### 5.40 1:J.j3306

- - li.. (!) 

### ca 

## 4.00 

# « li.. 

## y 

# = 1.6119x 2.00 Correlation coefficient (R)**/~** 

### : 0.99860 

## 0.000.00 2.00 4.00 6.00 Concentration(mg/L)



# o 

# o o C'fJ**~ ~**  

# kl

# ~

## Fig. 2-2 Calibration Curve Chromatogram 

## QUANTITATION Data File: 

## 9-MAR- 0 10:58 SamPle: 010 SamPle m/z: 

## 83.000 R.T.: 11'30" Standard m/z· 

## 45.000 R.T.: 11'00" 1.

## 0P9 *002 A: 555.7351 C: *001 R: 126
.0482 C: " 2.70mglL 

## STD R: 135.9386 R:4.0881 STD R: 136.4372 R:0.9238 O.540mglL 

## ID: ID: 10'59"

200 10'58" 200­

# ~

# ~ 3 

## 100­ 100 

# K

2" 

## A1'32" 11 12 11 12 *003 A: 1245.5172C: 5.40mglL



## STD 1=1: 

## 141.0457 R:8.8306 

## ID: 10'58"

200**~11'32"**

# ~ 

# ­ 100 

## J

# ~ 

# -. 11 12 -_

......_­ "-' 

# o (1)

### ~ 

## N 

# o

# @~

## Fig. 3 Chromatogram forAnalysis ofStockSolution 

## QUANTITATION Data rile:**9-M~R-** 

## 121 13:3121 SamPle: 

## 010 SamP 1

## eo m/z l 

## 83.121121121 R. T .**~** 

### 11/30" 

## Standard m/z' 

## 45.1211210 R. T.: 11/0121" 

## 1.0Pg *006**~:** 

## 795.9680 C:**~12112I5** 

## R: 718.5267 C: STD**~:** 

## 196.5254 R:4.0502 ! 

## STD R: 169.121450 R: 4. 251215 ID:

ID: 

## 11'30" 10'57" 1121 ' 57" 50**~11'** 

### 3130­ 

## 100 11210 

# \. \ 

# \
) 

# J\
11 

## 12 11 12



# c 

# c 

# o (1'l 

## .r-;: .'. . i**~** 

## '.-..>'

## ~ CJ

# ~

## Fig. 4-1 Chromatogram forAnalysis oftest sample (Condition-I) 

## QUANTITATION Data File: 9-MRR­ 0 14:46 SamPlE.': 010 SamplE.' m/z: 83.000 R. T.: 11/30" 

## Standard m/z: 45.0013 R.T.: 11/00" 1.**0P~** 

## 1'007 A: 158.8915 C: *008 R: 148.6672 

# c: waterphase 

### 101.1 water phase lot.2 

## sTD A: 73.7662 R:2.1540 

## 5TD R: 84.1788 R: 1. 

## 7661 

## ID: 

## ID: 50 50

100 100

50 50

11 12 11 12 1'009 A: 535.2990 

# c: *010 R: 634.6406 C: 1-0ctanolphase lot.l 1-0ctanolph(lse 

### 101.2 

## 5TD R: 165.6289 R:3.2319 

## sTD R: 167.6723 R:3.7850 

## ID: ID: 11/315'0 

## 50 

## 10/57" 11/31" 100 100

50 50

11 12 11 12


# o**~** 

# '-..d**~ ~** **~**

# ~ c

## Fig. 4-2 Chromatogram forAnalysis oftest sample (Condition-2)**QU~NTITATION** 

## Data File:**9-M~R-** 

# ° 15:54 

## SamPle: 010 

## SamP 1

## e 

## m/2: 83.000 R. T.: 11'30" 

## Standard m/2' 45.000 R.T.: 11'00" 1.0P9 +011 A: 124.7745 C: #012 A: 140.7385 C: waterphase 

## lot.! waterphase 101.2 

## STD**~:** 

## 142.9343 R:0.9846 

## STD A: 100.0212 R:1.2475 

## ID: ID: HI'57" 50 50

11 12 11 

## 12 *013 A:



## STD A: 

## ID: 60 40 20



# c c o 

# r:n**~** 

## C/l 

## 313.6966 C: *014**~:** 

## 271.8197 C: 133.7744 R:2.3450 1-0etanolphase lot.l 

## STD A: 148.5330 R:1.8300 1-0etanolphase lot.2 

## ID: 11'31" 10'S7" 11'31" 50

11 12 11 

## 12



# ,

__**~.**

# .J \... r-.J 

# -S\

01

### Fig. 4-3 

## Chromatogram forAnalysis oftest sample (Condition-3) 

## QURNTITRTION Data File:
. 9-MRR- 0 17:30 SamPle: 0H} SamPle m/z: 83.000 R.T.: 11'30" Standard 

## m/z, 45.131313 R.T.: 11'1313" 1. 13PS *1315

STD ID: R: R: 31. 3358 44. 1225 

# c: R:13.71132 waterphase lot.l


## 40

213

*017 R:

STn R: ID: 11 

## 197.13522 143.34313 C: R:1.3747 513 113'57"

1 J 

## 35" 12 l-Octanol 

### phase lot.l



## 40 11'31313

20



# c c 

# o C"; 

# ... en 11 12 *1315 R: 27.2835 

# c: waterphase lot2 

## STD R: 713.4418 R:13.3873 ID: 413 513

20 11'34" 11 

## 12 *1318 R: 223.13757 

# c: 1-0ctanolphase lot.2



## STD R: 181.13982 R:1.2318 ID: 11'313" 413 513

213

11 

## 12 

# 0ID



# --**~** 

# ..J\

## Table 1 Data table of stock solution ". Test substance Identification code
lot. 1 lot. 2 Volume of test solution Volume of analytical sample Injection volume Concentration of standard solution Peak area ratio of standard solution Peak area ratio of test substance in analytical sample

Concentration of test substance in analytical sample Residual rate ml ml III 

### mg/L
mg/L



# " 100 

### 10 2. 

### 70 4.0881 4.2505

2.81 100 100 

### 10 2. 

### 70 4.0881 4.0502

2.67 100 Conversion factor 1 1 

### Ratio of portion used for analysis 2500 2500 Absolute amount of test substance in analytical sample mg 0.0281 0.0267

Residual amount of test substance in test solution mg 70.2 66.9 Detection limited concentration mg/l 1.34 1.34 Concentration of stock solution mg/l 702 669**_~~ ",,_._._~_**  ••• ••**"~** •••••••_ ••••••• ••••••_ •• ,,,,_._.--,_.

___ ......... __•

_____ ._. ___ ._...... _ ••_ ................ _ ......._ ............. __ ._.......... ___••••_. ___ ...... _._ ••n ••

___••_._ ...... _______••

___ ._.... __ ..... __ ... ""

........ ___• 

### Average concentration of stock solution mg/l 685

,­ '-' 

# c o a"J



# ...... -J**~** 

# ....1\



# ,.,

## Table2 Data table of l-octanol phase

" 

### Testsubstance Identification code

Test condition 

## 1 condition-l condition-2 -._.

__. ___._._....__._.__...**_§!!!!Pl~l':Ip·** 

### ______..___..

# .l''._

...''

...__ 1

___._.._ .....__ 

### f.

____._._..___. __ 1

___._.__.....**~_** 

### ..._..___ Peak area ratio oftest substance in analytical sample

- 

# i 3.2319 3.7850 2.3450 1.8300 Concentration ofstandard solution mg/ll 2.70 2.10 2.70 2.70 Peak area ratio of standard solution 1 

### 4.0881 4.0881 4.0881 4.0881 Ir\iection volume J.ll 1 

### 1 1 1 Concentration oftest substance in analytical sample mg/I 

# i 2.13 2.50 1.55 1.21 Volume oftest solution ml 

### j 5 5 10 10 Conversion factor 

# ! 1 1 1 1 Ratio of portion used for analysis 

### j 125 125 250 250

Volume of analytical sample ml! 10 10 10 10 Detection limited concentration mg/l 

### 1 1.34 1.34 1.34 1.34 Residual rate 

# "1 100 100 100 100 Absolute amount oftest substance in analytical sample mg 

### 1 0.0213 0.0250 0.0155 0.0121 Residual amount oftest substance in testsolution mg**~** 

### 2.67 3.12 3.87 3.02 Concentration oftest substance in l-octanol phase mg/l 

## i 534 625 387 302



# o o (1)

# ~ condition-3



### ..._ . 

## .1

___......_.__ 2 

### 1.3747 1.2318

2.70 2.70 4.0881 4.0881 1 0.908 0.814

20 20 1 

### 1 500 500 10 10 1.34 1.34 100 tOO 0.00908 0.00814

4.54 4.07 227 203



# \....,**~** 

# W c

## Table 3 Data table ofwater phase

Test substance Identification code

Test condition 

# ! condition-1 condition-2 condition-3 

### 2 _
.....___

## ...J

_______..l_
..___.... 

## . __._..______..**_2.~~~** No_.

### ------.--~--- ............

# --.L--

...-.J-.-

...-.-..

## -.---.--?
.._______--=1'--_.



### Peak area ratio oftest substance in analytical sample 

## 1 2.1540 1.7661 1.2475 0.9846 0.7102 0.3873 Concentration of standard solution mg/L 

# ! 2.70 2.70 2.70 2.70 2.70 2.70 Peak area ratio of standard solution 

# ! 4.0881 4.0881 4.0881 4.0881 4.0881 4.0881 lr1iection volume J.I. 

### L 

## I 1 1 1 1 1 1 Concentration oHest substance in analytical sample mg/I! 1.42 1.17 0.824 0.650 0.469 0.256 Volume oftest solution mL 

### 1 35 35 30 30 20 20 Conversion factor 

### 1 1 

# ill 1 Ratio ofportion used for analysis 

## ! 11.7 11.7 10.0 10.0 6.67 6.67 Volume ofanalytical sample mL 

# I 10 10 10 10 10 10 Detection limited concentration mg/L 

### 1 0.0179 0.0179 0.0179 0.0179 0.0179 0.0179 Residual rate 

# "1 100 100 100 100 100 100 Absolute amount oftest substance in analytical sample mg 

### 1 0.0142 0.Q117 0.0082 0.0065 0.00469 0.00256 Residual amount oftest substance in testsolution mg 

# I 0.166 0.136 0.0824 0.0650 0.0313 0.0171

Concentration oftest substance in water phase mg/L 

# ! 4.76 3.90 2.75 2.17 1.56 0.853



# - '-' 

# o o \.,) 0') 

### -..,S:) 

# ... ..J:: CD

## Table 4 Data table ofPartition coefficient

... 

### Test substance Identification code Volume oftestvessel 44 mL Proportion ofsolution in test vessel:90.9" Added amount oftest substance 3.43 mg
Testcondition
. conditiorr-1 condition-2 condition--:3


## -.-...-...------.--.**San:a.P..t«:...t!4?~**

## __. ___. _____.._ .._._...._ ...._._..___________--...................---.-...-/...--
..-.!

..........-----.-.?.-.--

__..

## _..!

__. ___.._ .........

## ?

____.......

## _L
......_ .............g .........._. Volume ofphase (mL) l-octanol phasel 5 5 10 10 20 20

......

## ~.!ter**P.b.!!.~j** 35 35 30 30 20 20 i 

### Amount oftest substance (mg) l-octanol phasei 2.67 3.12 3.87 3.02 4.54 4.07 

### ._._!!
..

# ~.!.I!!:..p.has~J 0.166 0.136 0.0824 0.0650 0.0313 0.0171 Concentration oftest substance (mg/L) l-octanol phasel 534 625 387 302 227 203 i 

### water phase 

### 1 4.76 3.90 

### 2.75 2.17 1.56 0.853 Material balance (%) 

# I 82.7 95.2 115 90.1 133 119 

# Ipow I data 112 160 141 139 145 238 

## ! average data 

## i 136 140 192**~** 

### i Residual amount oftest substance in test solution lLogPow data 

### i 2.05 2.20 2.15 2.14 2.16 2.38 i I 

### Partition coefficient

### ~-.-- 

## ..**--.-.---.~.Y.~~l~~~--L-._-** 

## ...........--

__**2.:.'!~-.**

## __--_...._ .._

....._ ..**?.J.~** 

## ....__.._ ........._____.._?..2L_._. ltotal average Pow 

# i 156 i j 

### i LogPow 1 2.18 lstandard deviation (LogPow) 

# I 0.109 

### 1 LogPow[max]-LogPow[min] 

# i 0.327


# c 

# - (J") 

# ­ N 

# o 

# '"**~\**

## P-04-635 

## REPORT

Study Title 

## FRESH WATER ALGAL GROWTH INHIBITION TESTWITH

Studycompletion date 

### 28 October 

### 2005

Test Facility



### ,.- -,


### Laboratory Prolect Identification

## :~;):-T:~~ • Page 1of 36 •**~::;** 

### '. >_! 

### c.") CJ1



### 111111111111111111111111111111111111111111111111111111111111111111111111111 5 0 0 

### e 000 1 0 1 5 I S 

# -rD f**~oi** 

# - (P3S1. CONTENTS 1. CONTENTS

...........................................................................................................................2 2. STATEMENT OF GLP COMPLIANCE

..................................................................................4 

### 3. QUALITY ASSURANCE STATEMENT

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

### 5 

### 4. SUMMARy

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

### 6 5. INTRODUCTION

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

### 7 5.1. Preface

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

### 7 

### 5.2. Aim of the study

.......................................................................................,......................

### 7 5.3. Guidelines

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

### 7 5.4. Storaga and retention of records and materials

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

### 7 

### 5.5. Definitions

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

### 8 

### 6. MATERIALS AND METHODS

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

### 8 6.1.1. Test substance

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

### 8 6.1.2. Re1erenca substance

...........................................................................................8 6.1
..

### 3. PrelimInary data

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

### 9 6.2. Test System

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

### 9 6.3. Fresh water algae culture

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

### 9 6.4. Preparation oftest solutions

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

### 10 6.5. Range-finding test
........................................................................................................ 

### 10 6.6. First limit test
................................................................................................................

### 11. 6.7. Final limit 185t.

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

### 11 6.7.1. Test concentrations

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

### 11 6.7.2. Test procedures and conditions

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

### 11 6.7.3. Sampling for analysis of test concentrations

..................................................... 12 

### 6.7.4. Measurements

................................................................................................... 12 

### 6.7.5. Recording of cell densities

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

### 12 6.8. Interpretation

................................................................................................................ 12 

### 6.8.1. Data handling

..................................................................................................... 12 

### 6.8.2. Acceptabilfty of the test
...................................................................................... 

### 13 6.9. Ust of protocol davialions

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

### 13 7. RESULTS

............................................................................................................................. 14 

### 7.1. Ranga-finding test
........................................................................................................ 14 7.2. First limit test
................................................................................................................ 15 7.2.1. Measured test substance concentrations

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

### 15 7.2.2. Mean cell densities, inhibition of cell growth and reduction of growth rate
........ 

### 15 7.3. Final limit test.
.............................................................................................................. 

### 16 7.3.1
. Measured test substance concentrations

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

### 16 7.3.2. Mean cell densities

............................................................................................. 16 

### 7.3.3. Inhibition of 

### cell growth and reduction of growth rate
........................................ 

### 17 

### 7.3.4. Determination of effect concentrations

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

### 18 7.3.5. Experimental conditions

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

### 18 8. CONCLUSION

..................................................................................................................... 18 TABLES Table 1 Mean cell densities 

### (x10' cans/ml) during the range-finding 

### test
....................... 14 Table 2 Percentage reduction of growth rate and inhibition of total growth during the range-finding test
......................................................................................... 14 Table 

### 3 Mean cell densities (x 10 4 cells/ml) during the first limit te8t

... ........................... 15 Table 4 Percentage Inhibition of cell growth during the first limit test
............................. 

### 16 Table 

### 5 Percentage reduction of growth rate 

### at different time intervals during the first limit test
....................................................................................................... 

### 16 Table 

### 6 Mean cell densities (x 104 cells/ml) during the final limit test
............................. 

### 16 Table 

### 7 Percentage inhibition of cell growth during the limit test
.................................... 17 Table 

### 8 Percentage reduction of growth rate at different time intervals during the limit test
..............................................................................................................17 Table 9 Effect parameters
............................................................................................... 18 Table 10 pH levels recorded during tha limit test
.............................................................. 18
-Page2­Table 11 Table 12 Table 13 Table 14
FIGURES Figure 1 Figure 

### 2
APPENDICES APPENDIX I APPENDIX II APPENDIX 

### III Individual celf densities (1 S1 limit test)

................................................................. 19 Calculation of growth (area under growth curve) and growth rate (1 st limit test)

.................................................................................................................... 19 Individual cell densities (final 11mit test)

..............................................................20 Calculation of growth (area under growth curve) and growth rate (final limit test)

....................................................................................................................20
Growth curves at different concentrations of during the 1S\ limittest
................................................................................................... 15 Growth curves at different concentrations of during the final limittest
................................................................................................ 17
WORKSHEET DATA

.........................................................................................19 REFEAENCETEST

...........................................................................................21 ANALYTICAL REPORT

- Page 3­

### 2. STATEMENT OF GLP COMPLIANCE

The study described in this report has been correctly reported and was conducted In compliance with: The Organization for Economic Cooperation and Development (OECD) Good Laboratory Practice Guidelines (1997). 

### Which essentially conform to: The United States Food and Drug Administration Good Laboratory Practice Regulations. The United States Environmental Protection Agency Good Laboratory Practice Regulations. The sponsor Is responsible 

### for Good Laboratory Practice (GLP) compliance for all test substance information unless determined by**Study~** Head of In Vitro 

### &Environmental Toxicology

Date:

## .7.9.\.O.da::;,~:e.q 

# ...**~~~** 

# ............. Date:

........

# .u:?JR}.tOOf::.

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

-Page4­3. QUALITY ASSURANCE STATEMENT

This report was inspected by the Quality Assurance Unit to confirm that the methods and results accurately and completely reflect the raw data. 

### The dates of Quality Assurance Inspections are given be[ow. During the on-site process Inspections procedures applicable tothis type of study were Inspected.

Type of inspecUons 

### Phase/Process Start InspecUon date End Inspection date ReportIng date Study Protocol 03-Sep-04 03-Sep..()4 03-Sep-04 Amendment 1

### of protocol 03-May-05 03-May-05 03-May-OS Amendment 2 of protocol 30-Jun-OS 30-Jun-OS 3Q-Jun-05 Amendment 

### 3 of protocol 22-Jul-OS 22-Jul-05 22-Jul-05 Amendment 4 of protocol OS-Sep-05 OB-Sap-05 OS-Sep-05 Report 26-00t-05 26-0ct-05 26-0ct-05 

### Process Environmental toxicology 1S-Apr-05 OS-Aug-QS 22-Apr-oS 19-Aug-05 22-Apr-OS 19-Aug-oS Test substance handling Exposure Observalions/Measurements Analytical and phy6k:al 20-Jun-05 29-Jun·OS OS-Jul-06 chemistry 20-Sep-QS 26-Sap-05 27-Sep-05 Test substance handling Observations/Measuremants

Head of Quality Assurance

Date:

# 2.~ ..

# Q,.~~b~.200S

- Page 5·

### 4. SUMMARY 

### Selensstrum capricornutlJm, Fresh WaterAlgal Growth Inhibition Test with
The study procedures described in this report were based on the ISO Inlernational Standard 8692, 1989. In addition. the procedures were designed to meet the 1est methods and validity criteria ofthe EEC directive 

### 92169, Part C.3. 1992 and the OECD guideline No. 

### 201. 1984. The batch of tested was 

### a clear and colourless liquid with a purity 

### of 99.985%. was completely soluble in test medium at the ooncentrations tested and known to be extremelyvolatile. Consequently. measures were taken during this project to minimise loss of test substance by evaporation, 

### which irlcluded but were not limited to short preparation times and the use of airtight closed vessels. The project started with a ranoe-findinQ test eXPOSing exponentially growing algal cultures to a blank-control and nominal concentrations 

### of0.1, 1.0, 10 and 100 mgll. No significant effects were observed on algal growth inhibition and growth rate reduction up to

### a nominal test concentration of 

### 100 mg/l. Samples taken from nominal 

### 10and 

### 100 mg/I were analysed, which showed mean start concentrations of 1.0 and 14.6

## mgn, respectively. These concentrations decreased by approximately

### 80 to 

### 90"10 below initial during the test period despite the fact thattest vessels were airtightly closed. The project was continued with a limit test applying an adapted preparation method. Exponentially growing algal cultures were exposed to a blank-control and a nominal concentration 

### of 147 mg/1. An amount of 100pi

## was pipetted directly into 1 liter (nominal 147 mg/!) 

### ofstirring algal growth medium. The vessel was immediatelyclosed and the content magnetically stirred for 1 minute. Then, individual replicate vessels were filled with the solution. algae were added, samples were taken and the incubation started in completely closed vessels. The total test period was 72 hours and the initial algal cell density was 10"celis/mI. Analysis at the start 

### ofthe limit test showed a mean measured concentration 

### of 57mgll in samples taken from nominally 147

### mgII. The measured concentration decreased to 6.3 mgll after24 hours and to 

### 2.1 mgll after 72 hours of exposure. The Time Weight 

### Average (1W

### A) concentration was calculated to correspond to 

### 8.7 mgll. As the initial concentration was well below 100 mg/I in the first limit study it was decided to repeat this study. In the final limit study an amount of 175

### pi was pipetted into 1 liter of stirring algal growth medium (nominal 257 

### mgII). Further test procedures were comparable to the first limit study, except that headspace in the test vessels was reduced by 50%. Analysis at the start of

### thefinal limit test showed a mean measured concentration of 213 mg/l in samples taken from nominally 257 mg/l. The measured concentration decreased to 13 mg/l after 

### 24 hours and to 

### 6.3 mg/I after 72 hours of exposure. TheTime Weight Average (TWA) concentration was calculated to correspond to 24 mglL The study met the acceptability criteria prescribed bythe protocol and was considered valid. No significant Inhibition of cell growth Of reduction of growth rate was recorded at nominal concentrations up 10 257 mg/I, corresponding to a measured Initial concentration of 213 mg/I or aTWA concentration of 24 mgl1 (NOEC). The 

### ECso for cell growth inhibition and growth rate reduction exceeded 

### a measured initial concentration of 213 mgll 

### or aTWA concentration of 

### 24 mg/l

.

• Page 6·5. INTRODUCTION 

### 5.1. Preface Sponsor

Study Monitor Test Facility

Study Director Technical Coordinator 

### Principal Scientist Study Plan 

### Start 23 May 2005 Completion 

### 16 September 

### 2005 

### 5.2. Aim ofthe study The purpose of the study was to evaluate the test substance for Its ability to generate toxic effects in 

### Selenastrum caprlcomutum during an exposure period of 

### 72 hours and, 

### it possible, to determine the ECso for 

### both inhibition of cell growth and reduction of growth rate forthe 72-hour period. 

### 5.3. Guidelines The study procedures described In this report were based on the ISO Intemational Standard 

### 8692: 'Water quality
- Fresh water algal growth inhibition test with 

### Scenooesmus subspicatus and 

### Se/enastrum capricomututrl', First edition, 

### 15 NOV9mber 

### 1989. In addition, the procedures were designed to meet the test methods 

### andvalidity criteria prescribed by the following guidelines: • European Economic Community (EEC). EEC directive 

### 92169, Part C: Methods for the determination of ecotoxlcity. Publication No. 

### L3SS, December 

### 1992, C-3: "Algal Inhibition 

### Test". • Organization for Economic Co-operation and Development (DECO), OECD guideline for Testing of Chemicals. guideline No. 

### 201: "Algae, Growth Inhibition Test", Adopted June 7. 

### 1964. 5.4. Storage and retention 

## or records and materials Records and materials pertaining to the study including protocol. raw 

### data, specimens (except specimens requiring refrigeration orfreezing) and the final report are retained 

### in the archives for a period of a11east 

### 10

### years after finalization of the report. After this period, the sponsorwill be contacted to determine whether raw 

### data and specimens should be returned to them. retained or destroyed on their behalf. Those specimens requiring refrigeration orfreezing will be retained by for 

### as long 

### as U1e quality of the speoimens permits evaluation but no longerthan three months afterfinalization of the report.
.

• Page 7·NOTOX will retain a test substance sample until the expiry date, but no longerthan 10 years afterfinalization ofthe report. After this period the sample 

### will be destroyed. 

### 5.5. Definitions Cell density is the number of cells per millilitre. Total growth orbiomass Is defined as the increase in total cell density overthetest period. It is derived from the area under the growth curve in a linear plot. The EBCSO is the concentration of test substance that results in a 50% inhibition of total cell growth relative tothe control. Growth rate isthe increase in cell density per unit time. It is derived from the slope of the growth curve in a logarithmic plot. Following from the mathematical nature of exponential growth, the measure of the specific growtl1 rate is preferable over biomass. The 

### ERGsO is the concentration of test substance that results 

### in a50% reduction In growth rate relative to the control. No Observed 

### Effect Concentration (HOEC) is the highest concentration tasted at which the measured parameter(s) show(s) no significant effect on algal growth relative to control values.


### 6. MATERIALS AND METHODS 

### 6.1.1. Test substance Identification Structure
Molecularformula Molecularweight CAS Number Description Clear colourless liquid Batch Purity 99.985% Test substance storage In refrigerator In the dark Stability 

### under storage conditions Stable Expiry date 

### 28 February 

### 2006 Specific Gravity 

### 1.47 Stability 

### in water Not indicated 

### 6.1.2. Reference substance This report includes the results of the most recent reference test with potassium dichromate (Appendix II).

- Page 8·

### 6.1.3. Preliminary data The water solubilitv of at 

### 20.1 ± 

### 0.5"C is 

### 0.805 gil using the flask method Further essential information obtained from physico-chemical studies performed at were the vapour pressure being 

### 31 kPa at 

### 25°G. freezing point 

### <-81 "C. boiling point 56°C and a log 

### Koc of 2..54. The Log Powof was 2.18 according to information supplied bythe sponsor. From these data it can 

### be concluded thatthe

. tast substance will evaporate rapidly at room temperature as pure substance but also from 3 l aQueous 

### solutions (calculated Henry coefficient is 

### 7.24.103 Pa m mor ). concentrations showed to 

### be stable during treezing and thawing of samples 

### in 

### a deep-freeze 

### 6.2. Test System Species 

### Se/enastrumcapricomurum, strain: range-finding test and 181 limittest NIVA CHL 1; final limit test: CCAP 

### 27814. Source In-house laboratory culture. Reason for selection This system is an unicellular algal species sensitive to toxic substances in the aquatiC ecosystem and has been selected 

### as an internationally accepted species. 6.S. Fresh 

### water aJgae culture Stock culture Algae stock cultures were started 

### by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated 

### and exposed to light in 

### a climate room at atemperature of 

### 23 ±2"C. Light intensity 

### 60to 

### 120pEtm'ls when 

### measured in the photosynthetically effective wavelength range of

### 400 to 700nm. Stock culture medium 

### M1; formulated using Milli·Ro water (tap-water purified by reverse osmosis; Mlilipore 

### Corp.• Bedford, Mass., 

### USA) with the following composition: NaNOa KzHPO. 500 40 mgII 

### m\il MgSo •.**7H~** 76 

### m\il NII2CO".10H.O 54 mgll CeHaOr.HaO 6 mgll**NH~** 330 mgll CaCI2.HzO 

## se mgll**CeH5Feo,~O** 6 mg/l H3BOa 2.9 mg/l t.AnClz .4HrO 1.81 mgll ZrICfz (1.11 mg/l CuSO

..**5H~O** (I,OS mg/l**(NH..)G~OM-4HtO** 0.018 mgt1 Pre-cuRure 3 days beforethe start ofthe test, cells from the algal • stock cuHure were inoculated in cuHure medium ata cell density 

### of 2.10" celis/mi. The pre-culture was maintained under ttie same conditions as used in the test. The cell denSity was measured immediately before use.



### • Page 9·Pre-culture medium M2; according tothe ISO-Standard "Algal growth inhibition test" Nov. 

### 1989; formulated using MiIIi..Q water (tap water purified 

### by reverse osmosis 

### (MiIIi-RO) and subsequently passed over activated carbon and ion-exchange cartridges: MiIIi-Q water; Millipore Corp., 

### Bedford, Mass., 

### USA) preventing precipitation and with ttlefollowing composition:**N~** 15 mgll MgC!••**6H~** CaClr,.2HaO 12 18 mgll mgll MgS04.7HJO I<liJ!P04 15 1.6 mgll mgll**F~6HP** 80 pg/l**NIbEDTA.2H~** 100 pgll**~o.** 185 pgll**MnCb.4H~ ZnC~**  415**~** 

### #0/1 pgII COCI:.6H.O 1.5 pgII CuClr,.2HP 0.01 pg/l**Na,.1iAo04.2H~** 7 pgII NaHCOs 100 mgll Hardness (CIl+Mg) 0.24 mmof.ll (24 mg CaCOJl) pH 8.3:1;0.2 6.4. Preparation of

### testsolutions The standard test procedures required generation of test solutions. which contained completely dissolvedtestsubs1ance concentrations or stable and homogeneous mixtures or dispersions. Thetesting of concentrations that would disturb the 

### test system was prevented as much as possible (e.g. film ofthe test substance on the water surface). The batch of tested was a clear and colourless Ilquld with a purity of 

### 99.985%. was completely soluble in test medium 

### atthe concentrations tested and known to be extremelyvolatile. Preparation 

### oftest solutions forthe range-finding test started with weighed amounts oftest substance that 

### were quantitatively added to volumetric flasks to provide test solutions of nominally 

### 10 and 

### 100 mg/l. The test solutions were magnetically stirred for 

### 2-5 minutes. Care was taken that during preparation as little as possible evaporation occurred (weighing and preparation In closed vessels which already contained a small volume of test medium, short preparation period). The lower test concentrations were prepared by dilution of the test solution of 10 mgIJ. For the first limit test atarget concentration 

### of 100 mg/1 

### was prepared by pipetting an amount of 100 pi directly into 1 liter (nominal 147 mgII)

# 0' stirring algal growth medium. The pipetted volume forthe final limit test was 175 pi per liter (nominal 257 mgn). The vessels used for preparing thetest solutions were immediately closed and the content magnetically stirred for 1 minute 

### (first limit) or 5 minutes (final test). Directly afterthe short stirring period. volumes 

### of 50 ml 

### (first limit 

### test) or 75 ml (final test) were added to each replicate ofthe respective test concentration. Hence. in the final study the headspace ofthe test vessels was reduced by 50% to further minimize loss of test substance ooncentration byevaporation. Subsequel'ltly, 1 mlof an algal suspension was added to each replicate providing a cell density of 1 Q4 cells/ml, samples were taken and the incubation started. The final test solutions were 

### art clear and colourless. 

### 6.5. Range-finding 

### test A range-finding test was performed to provide information about the range of ooncentrations to be used in the final test Test procedure and conditions were similarto those applied in the flnal test with the following exceptions: Exponentially growing algal cultures were exposed to a range of 0.1 to 100 mgllincreasing by a factor of 10 and to a blank-oontrol. Three replicates were tested per concentration and three

• Page 10·replicates in the control group. One extratest vessel per concentration without algae was used as background for the determination ofthe algal cell density 

### at each time interval. 

### pH was only measured in the control and the highest test concentration. Duplicate samples for possible analysis were taken from all concentrations, but the control. The NaHCOsconcentration in the test medium was 50 mgll and the vessels were filled with 50 ml solution. 

### 6.6. First IImhtest A first limit test

### was performed with algae exposed to a nominal concentration of 147 mgJI, prepared bydirect addition of 100pi of the puretest SubstaJ'1C9 to a liter

### test medium. A blankcontrol was included and test procedures and conditions were similartothose applied during the final test exceptforthe factthat the NaHCOs concentratlon In the test medium was 50 mg/l and that vessels were filled with 50 ml 

### testsolution. 

### 6.7. Anal limittest 6.7.1. Testconcentrations 175

## JM, or nominal 257 mg/l, expected to result in a start conoentratlon of 

### at least 100 mgJI, I.e. the regulatory limit concentration. Controls Test medium without test substance or other additives (blank-control)

. Replicates 

### 6 replicates of the blank-control and test concentration. 

### 2 replicates of the test ooncentration without algae. 1 extra replicate ofthe blank-control andthe test concentration for sampling after 24 hours of exposure. 6.7.2. Test procedures and conditions Test duration 72 hours Testtype Static Testvessels 100 ml, all-glass, containing 75 ml of test solution, airtight closed with a screw cap. Medium 

### M2 Cell density 

### An Initial cell density of 1 x 1()4 celis/mi. Illumination Continuously using TLD-Iamps of the type 'Cool-white' of 30 Watt, with a Ught intensity within the range of 67 to 

### 82 

### J,LE.m·2

### .s· 1

. Incubation Vessels were distributed at random in the Incubator. During incubation the algal cells ware kept in suspension by continuous shaking•

• Page 11 ­

### 6.7.3. Sampling for analysis of

### test concentrations 

### During the final limittest duplicate samples were taken from the blank-control and the limit concentration. Samples were transferred to headspace-vlals that were immediately capped airtightty. The method 

### ofanalysis Is described in the appended Analytical Report (Appendix III).

. 

# . Frequency 

### att=O 

### h (without algae only). t=24 h and t=72 h 

### Volume 10ml Storage Samples taken at the start and after 24 

### hours were stored in 

### a freezer until analysis. Compliance with the QualHy crHena regarding maintenance of 

### actual concentrations was demonstrated by running a 

### test vessel without algae and duplicate samples for

### analysis were taken from the limit concentration at the start, after 24 

### hours ofexposure and at the end ofthe test period. 6.7.4. Measurements 

### pH At the beginning and at the end 

### ofthe test. The 

### pH of the solutions should pr9ferably not deviate 

### by more than 

### 1.5 units 

### during the test. Temperature of 

### medium Continuously In 

### a temperature control vessel. 6.7.5. Recording of cell densities 

### Atthe beginning of 

### the test, cells were counted using 

### a microscope and 

### a counting chamber. Thereafter

### cell densities were determined 

### by spectrophotometric measurement 

### ofsamples at 720 nm using a Varian CaIY 50 single beam spectrophotometer with immersion probe 

### (pathlength =20 

### mm); Varian Nederland B.V., Houten, The Netherlands. applying Bio Pack version 2.00(26) 

### software. Algal medium was used as blank. To prevent additional loss of 

### test substance during spectrophotometric measurement, samples of 

### -2 ml were taken from the closed test vessels through the septum in 

### the screw cap using a syringe 

### and needle. Samples ware transferred into 

### a eweUe and then algal density was measured. 6.8. Interpretation 6.8.1. Data handling 

### Calibration curve Quantification of cell 

### densities was based on a 

### calibration curve. Cell 

### density was 

### ploUed versus extinction using spectrophotometric measurements of a minimum 

### ofsix dilutions of an algal suspension with different cell densities. The calibration curve 

### was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period. Comparison of areas under the growth curves The area below the growth curve was calculated using the formula:

Where: A :;; area 

### No=: nominal numberof cellslml at the start of 

### the test N

1

# = measured number of cells/ml at

### t, 

### Nn;; measured number of cells/ml at tn t,= 

### time of

### first measurements after beginning of 

### the test**~=** 

### time 

### of nih measurement after beginnIng otthe 1ast

- Page 12­The percentage inhibition of cell growth at each test concentration**(~)** was calculated using the following formula: IT= 

### lie -A,x100 

### Ac Where: 

### Ao

# = area below the growth curve obtained 

### in the control 

### AT=area below the growth curve at each test substance concentration Growth inhibition was calculated for the total period of 72h. Comoarison of growth rates The average specific growth rate 

### (.ti) for exponentially growing cultures was calculated as:**InNn-ln~** p=**tn-~** The average growth rate at each test substance concentration was then compared to the control' value andthe percentage reduction in growth rate was calculated. Determination of the average exposure concentrations The average exposure concentrations were calculated as:

. 24X'/CI-oXC,.w;24ax.jC,.".XCtc72 being the TimeWeightAverage (TWA) of the concentrations of I measured in the samples taken atthe start 

### (Ct.o), after 24 hours 

### (Cw.4) and the end ofthe test (C1 ,,72). Determination of 

## the NOEC and calculation 

### of the 

## ECso For determination of the NOEC and the**E~** the approaches recommended in the DECD guideline 

### (201, adopted 7 June 1984) were used. Statistical analysis of the data was not needed as the effects recorded were not significant. No EC50-values could be calculated because the test substance proved to be non-toxic 

### (ECso > maximum concentration tested). 

### 0.8.2. Acceptability ofthetest 1. In the controls. cell density increased by an average factor of > 16 within three days. 2. Further, test conditions (temperature and pH) remaJned within the ranges prescribed bythe guideline, except for the pH. The pH increased by more than 1.5 unit. but 

### this was caused by the fact that test vessels were air tightly closed. 

### 6.9. List of

### protocol deviations 1. The pH in the range-finding test and the two limit tests increased above a value of 9.0 and/or with more than 1.5 units. Evaluation: The observed increase was due tothe fact that test vessels were completely closed and consequently gas exchange was reduced in comparison to norma! open systems. Increasing 

### 1he NaHCO,concentration in the medium prevented an Increase In pH of more than 1.5 units but could not maintain the value below 9.0. The study Integritywas not adversely affected bythe deviation.

- Page 13­7. RESULTS 7.1. Rang&-nndlng test The mean cell densities measured during the range-finding test are presented in Table 1. Table. 

### 2 presents the percentages growth inhlbltJon and growth rate reduction perconcentrat[on. No biologically significanteffects were observed on algal growth inhibition and growth rate reduction up to a nominal test concentration of 100 mgll. Samples taken from nominal 1 0 and 

### 100 mg/I were analysed. which showed mean start concentrations of 

### 1.0 and 

### 14.6 mg/l, respectively. These concentrations decreased byapproximately 80to 90% below initial during the test period despite the fact ,hat test vessels were airtightly closed (seeTable 

### 2 of the appended Analytical report). All test conditions were maintained within the limits prescribed bythe protocol. except forthe pH that increased above 9.0, which was likelyto be caused bythe fact that test vessels were closed. Table 1 Mean cell densities (x104 cellslml) during the range-finding test Concentration Exposuretime (hours)**~I(mg/l)** 0 24 48 72 ntro! 1.0 6.2 18.4 43.0 0.1 1.0 5.5 13.5 37.9 1.0 1.0 4.B 14.9 32.2 10 1.0 3.0 13.5 41.5 100 1.0 

### 4.6 15.3 40.9 Table 2 Percentage reduction ofgrowth rate and inhibition oftotal growth during the range-finding 

### test ConcentraUon Cell growth (0-72 hra) Mean growth rate Nominal (mgl'l) Mean area (A) Inhibition**(~I** II(0-72 hrs) 

### Reduotion (%'1 Blank-control 1047.24 0.05224 0.1 851.24 18.7 0.05025 3.8 1.0 798.80 23.7 0.04819 7.8 10 834.48 20.3 0.05173 1.0 100 907.64 13.3 0.06153 1.4 Dueto the extreme volatile charactar of the test substance, 

### it was not possible 

### to start atthe intended nominal concentration with the method of preparation used in the present rangerlnding test (see paragraph 

### 6.4) and 

### to keep concentrations stable during exposure. Therefore, it was decided to adapt the preparation procedure for the final test as to be able to start at an actual exposure concentration close 

### 10 100 mglL Forthis purpose, an amount of 

### 100pi was plpetted directly into 

### 1 liter (nominal 

### 147 mg/l) ot stirring algal growth medium. The vessel was immediately closed and the content magneticallystirred for 1 minute. Immediately thereafter, individual replicate vessels were filled with test solution, algaewere added, samples were taken and 

### the incubation started 

### in completely closed vessels. All 

### within the shortest time possible.



### - Page 14·7.2. First limntest 7.2.1. Measured teat sub,lance concentrations The results of analysis of the samples taken duringthe first limit test are described in Table 

### :3 of the appended Analytical Report. Analysis at the start of the test showed 

### a mean measured concentration of 57mg/l in samples taken from nominally 147 mgll. The measured concentration decreased to 6.3 mg/l after24 hours and to 2.1 mgJI after

### 72 hours of exposure. The Time WeightAverage (TWA) concentration 

## was calculated to correspond 

### to 8.7 mgJI. 7.2.2. Mean cell denslUes, Inhibition

### 01 cell growth and reduction ofgrowth rate Table 3 shows mean cell densities measured at 24-hour Intervals atthe limit concentration of The respective growth curves are shown In Figure 1 (seeAppendix I for the cell densities per replicate). Table 4 shows the calculation of !he percentages of inhibition andTable 5 shows the percentages 01 growth rate reduction at different time Intervals (see Appendix I for the areas of cell growth and values of growth rate 

### per replicate), No significant differences were recorded between the values for cell growth or growth rate between the limittest concentration and the blank-control. However, 

### as the initial measured concentration was only 57 mg/l. andthus stili not at the required 100 mgJI. it was decided to perform a second limit test In the second Imittest a higher nominal conoentretion was prepared as to be able to startthe

### test atthe required minimum concentration 

### of 100 mgll. 

### All test conditions were maintained within the limits prescribed by the protOCOl. except for the pH that inoreased with more than 1.5 units to avalue of 10.4

### atthe end 

### ofthetest. The pH increase was probablycaused bythe fact that test vessels were closed hampering the CO2 exchange. Table 3 Mean cell densities (x 10 4 cell'lml)duringthefirst limit

### test ConcentraUon (mgII) Nominal/initial Expaauretime (hour.) 

### 0 24 48 72 Blank-control 1.0 3.9 14.8 36.9 147 I 

### 57 1.0 4.5 12.5 30.9
40 jt' 

# /x / ,--' .' 

# /,./ 

# /--,­ /.x" ./


# ... .'**--,-~** 5 

### oo 24 nomlnBl (mgll)

-·.··147

Figure 1 Growth curves atdifferentconcentrations of during the 1-' limittest

• Page 15·Table

### 4 Percentage Inhibition ofcell growth during the

### first limittest ConcenbaHon(mgn) Cell growth (0-72 hrs)

Nominal/Initial 

### Mean a.... (A) InhibHion (%) Blank-control 

### 830.94

147 I 57 717.80 13.6 TableS Percentage reducUon of growth rate

### atdifferenttime Intervals duringthe first limittest Concentration (mgn) 

### Mean growth rate

Nominai/lnlDal 11(0-24 hra)· Reduction (%) II(0-48 hl'8) RedUction (%) II(0-72 hrs) Reduction (%) Blank-<:ontrol 0.05643 0.05612 0.05011

147 I 57 0.06148 -9.0 0.05259 

### 6.3 0.04765 4.9
7.3. Final limittest 7.3.1. Measured 

### test substance concentrations 

### The results of analysis of

### the samples taken during the final limit

### test are described in 

### Table 4 of. 1he appended Ana!ytical Report. Analysis atthe start 

### of the 

### test showed a mean measured concentration of213 mgll in samples taken from nominally 257 mg/l. Hence, this time a much higher recoverywas achieved in the samples taken atthe start of exposure. The measured concentration decreased to 13 mgll after 24 hours and to 6.3 mgll after 72 hours of exposure. The Time Weight Average (TWA) concentration was calculated to correspond to24 mgJI. 7.3.2. Mean 

### cell denslUes 

### Tabla 6 shows mean cell densities measured at 24-hour intelVals atthe limit ooncentration of The respective growth ourves are shown in Figure 2 (see Appendix Ifor the cell densities per replicate). Table 6 Mean cell densities (x 1

## rt cellS/ml) 

### during thefinal limit

### test

Concentration (mgn) 

### Exposuretime 

### (hours)

Nominal/Initial 0 24 48 72

Blank-control 1.0 3.1 11.3 33.4 257 J 213 1.0 3.3 12.0 34.7

• Page 16­40 35 

# .? Concentration nomInal (mlJll)

# ~f' 

# / -+-Blankcontrol 

# /**~/ ~**  5 

### o**~**

o 24 48 72 Exposure time (hours) Figure 2 Growth curves at different concentrations of during the finalllmHtest 7.3.3. Inhibition of cell growth and reduC1lon ofgrowth rate 

### Tabla 7 shows the calculation of 

### the percentages of inhibition and Table 8 shows the percentages of 

### growth rate reduction at different time intervals (see Appendix Ifor the areas of 

### oell growth and values of 

### growth rate per replicate). No significant differences were recorded between the values for cell growth orgrowth rate between the 

### test concentration and the blank.-control. Table 7 Percen1age inhibition of cell growth during thefinal limittest Concentration (mgll) Cell growth (0-72 hrs) Nominal/Initial Mean area (A) Inhibition (%) Blank-control 686.70

257 , 213 722.50 -52 Table 8 Percentage reduction of growth rate at differenttime Intervals during the final limittest Concentration (mgll) Mean growth rate Nominal/Initial p(D-24 hrs) Reduction (%) p (0-48 hrs) Reduction (%) p (0-72 hrs) Reduction(%) Blank·control 0.04742 0.05044 0.04869

257 I 213 0.04935 -4.1 0.05170 -2.5 0.04925 -1.1

- Page 17­

### 7.3.4. Oetennlnatlon of

### effect concentrations Table 9 

### shows the effect 

### parameters based on 

### nominal and Initial 

### measured concentrations. 

### Table9 Effect 

### parameters Parameter conc.nlndion lmalll Nominal I Initial NOEse 257 I 213 72h-EeC50 >257 I >213 NOE"C 257 1213 72h-ERC50 >257 I >213


### 7.3.5. Experimental 

### conditions Table 10 

### shows the pH 

### recorded at the beglnning and the end of the test. The pH was within the limits prescribed by the protocol (6.0-9.0, 

### preferably not varying by more 

### than 1.5 

### unit) 

### atthe start of 

### exposure, but exceeded the upper 

### limit al

### theend of the test due to the fact

### that test vessels 

### were airtight closed. 

### The temperature of

### the

### test medium 

### was 22.B"C at

### thestartof 

### the test. During 

### the exposure 

### period the temperature measured in the incubator

### was maintained between 22.1 and 23.6°C. 

### Temperature remained 

### within the limits 

### prescribed bythe 

### protocol (21-25°C, 

### constant within 2°C). 

### Table 10 pH levels recorded during 1I1e

### final limittest

Concentration (mg/l) Exposur.time (hours) Nominal/Initial 0 72 

### Blank-control 8.4 9.8

257 I 213 8.4 9.9


### 8. CONCLUSION Under the 

### conditions of the.present 

### study with 

### Selenastrum csprlcornutum, no Significant 

### Inhibition of 

### cell growth or reduction of growth 

### rate was recorded 

### at nominal concentrations up to 

### 257 

### mgJI, corresponding 

### to a measured initial 

### concentration of 213 mwl or aTWA concentration 

### of 24 

### mgJI (NOEC). The EC50 

### for cell growth inhibition and growth rata reduction exceeded a measured initial concentration of 

### 213 mg/I 

### or aTWA concentration 

### of 24 mg/I.

-PagelS­APPENDIX I WORKSHEET DATA
Table 11 Individual cell densities (1 111 limittest) Number of Inoculated cells aU..o: 1 x 10 cells/ml Concentration
Nominal (mgll) Vessel number Exposure Ome (hours)
0 24 48 72 Blank-corrtrol 1 1.00 3.94 14.44 35.53 2 1.00 3.71 15.32 38.22 3 1.00 4.14 14.82 38.44 4 1.00 3.79 1421 36.38 5 1.00 3.72 15.00 36.81 IS 1.00 3.96 14.98 38.03 147 1 1.00 4.36 12.63 31.29 2 1.00 6.45 13.35 31.95 3 1.00 4.08 11.94 32.27 4 1.00 3.93 12.18 29.69 S 1.00 4.17 13.30 31.09 6 1.00 3.72 11.60 29.19

Table 12 Calculation of growth (area under growth curve) and growth rate (1l11Umlt test) V8&&81 Concenntfon lvea(A) Qrowth rate Qrowth Inhlb. (%) GrowthrateAlductlon (%) number 

# , Nominal (mgll) Q.48hrs O·72hra 0-2.4 hra G-72hrs o.72hrs Q.48brs 0-24 hra Blank-oontrol 1 8IJ7.48 0.05713 0.0S5S3 0.04959 2 856.36 0.05463 0.05686 0.05060 3 0.05920 856.32 0.05617 0.05068 4 806.56 0.05552 0.05529 0.04992 

# . Ii 831.00 0.05474 0.0600S 6 826.92 0.05734

# ~:~~e: 0.04978 Mean 831 0.05643 0.05612 0.05011 147 1 0.04782 13 -9 6 5 2

# :!~ 0.077 0.05399 0.04811 4 ·38 4 4

# ~O~~ 3 711.72 O. 0.05166 0.04825 14 ·4 8 4 4 682.92 O. 0.05208 0.04709 18 ·1 7 6 5 732.36 O. 0.05391 0.04773 12 ·5 4 5 6 657.96 0.0547 0.05105 0.04686 3 21 9 6

- Page 19­APPENDIX I WORKSHEET DATA (Continued)
Table 13 Individual cell densities (final limittest) Concentration 

### v..... Exposuretime(hours) number Nominal(mgll) 0 24 48 72 Blank-oontrol 

# , 1.00 3.47 11.43 34.62 2 1.00 3.40 12.77 37.09 3 1.00 2.91 12.00 34.32 4 1.00 2.85 10.23 30.34 5 1.00 3.09 10.89 32.13 6 1.00 2.95 10.44 31.79 257 1 1.00 3.25 11.85 35.46 2 1.00 2.79 11.38 34.02 3 1.00 3.51 11.44 34.33 4 1.00 3.79 11.60 33.13 5 1.00 3.20 12.21 34.87 6 1.00 3.16 13.39 36.50

Table 14 Calculation ofgrowth <area under growth curve) and growth rate (final limit

### test) Concentration
Nominal (mgll) Vessel number Area (A) Growth rats Growth Inhlb. (%) GrowU1 rate reduction (%)
O-72nra 0-24hrs 0-48 hre 0-72 hra 0-72hrs Q..24hrs 0-48hrs O-72hrs Blank-control 1 713.04 0.05184 0.05076 0.04923 2 m.16 0.05099 0.05306 0.06019 3 709.68 0.04451 0.05177 0.04911 4 620.40 0.04508 0.04844 0.04740 5 661.08 0.04701 0.04975 0.04819 6 642.84 0.04508 0.04887 0.04804 Mean 687 0.04742 0.05044 0.04869 257 1 7ZT.92 0.04911 0.05151 0.04956 -6 -4 -2 ·2 2 688.32 0.04275 0.05068 0.04899 0 10 

## a -1 3 710.76 0.05232 0.05077 0.04911 -4 -10 ·1 

# ., 4 708.92 0.05552 0.05100 0.04862 -3 ·17 ·1 0 5 725.88 0.04846 0.05213 0.04925 -6 -2 -3 -1 6 775.20 0.04794 0.05405 0.04998 

# -'3 -1 ·7 -3

- Page 20­APPENDIX II REFERENCE TEST 

### Selanastrum caprlcomutum. strain: NIVA CHL 1. Fresh water algal growth inhibition test with potassium dichromate Start of first exposure: 14 March 2005 Completion last exposure: 17 March 2005 The study procedures described in this report were based on the EEC Directive 

### 92169, Publication No. 

### l383 Part 

### C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989. This reference test was carried out to check the sensitivity ofthe test system used by to Potassium dichromate (Merck, Art. 4864, Batch K28974764). Algae were exposed for 

### a period of 72 hours to K2Cr2()., (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a blank-control. The initial cell density was 1.0

### x 104 cells/ml. Results: Overview of 

### % inhibition in cell growth and % reduction of growth rate in the reference test:

Concentration K2Cr207 (mgll) Total cell growth: Mean Area Inhibition O-72h % Growth rate: Interval Reduction 0-72h % Blank-control 1508.80 0.06087 0.18 1404.52 6.9 0.05964 2.0 0.32 1376.44 8.8 0.05923 2.7 0.56 1374.08 8.9 0.05936 2.5 1.0 1127.88 25.2 0.05575 8.4 1.8 560.24 62.9 0.04218 30.7 3.2 217.20 85.6 0.02301 62.2 Underthe conditions of the reference study with 

### $alanastrum capricomutum, potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 1.0 mgll and higher. The EC60for cell growth inhibition (EeC50: 0-72h) was 1.5 mg/l with a 95% confidence interval ranging from 1.0to 2.2 mgll. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/l. Hence, the ESC50: 0-72h for the present batch corresponds with this range when taking into consideration the confidence interval. The EC50for growth rate reduction (ERCSO: 0-72h) was 2.6 mgll with a 95% confidence interval ranging from 2.0 to 3.2 mgll. The historica! ranges for growth rate reduction lie between 0.82 and 2.3 mgll. Hence, the ER C50: Q-72h for the present batch corresponds with this range when taking into consideration the confidence intervaL The protocol, raw data and report of this study are kept in the archives. The test described above was performed under GLP conditions with a QA-check.

- Page 21 ­

## APPENDIX III

FRESH WATER ALGAL GROWTH INHIBITION TEST WITH
DETERMINATION OF THE CONCENTRA"nONS

Study 

### completion 

### date

October28, 2005

Laboratory Profect Identification

- Page 1 of 15 ­

### 4 1. CONTENTS 1. CONTENTS

...........................................................................................................................2 

### 2. REPORT APPROVAL.

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

### 3 

### 3. INTRODUCTION

....................................................................................................................4 3.1. Preface

...................................:.......................................................................................4 3.2. 

### Aim ofthe study

................................................ n ............................................... < ............ 

### 4. MATERIALS AND METHODS

...............................................................................................4 . 4.1. Reagents

........................................................................................................................4 4.2. Samples

..........................................................................................................................4 

### 4.3. Analysis

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

### 5 4.3.1. Analytical method

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

### 5 4.3.2. Calibration

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

### 5 4.3.3. Injections

..............................................................................................................5 4.3.4. Data acquisition and data processing

..................................................................6 4.3.5. Procedural recovery samples

..............................................................................6 

### 4.4. Interpretation

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

### 6 4.4.1. Calculations

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

### 6 4:4.2. Formulas

..............................................................................................................6 5. RESULTS

...............................................................................................................................8 5.1. Calibration curves...........................................................................................................8 5.2. Samples

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

### 8 5.2.1. Procedural recovery samples

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

### 8 5.2.2. Test samples

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

### 8
TABLES Table 

### 1 Procedural 

### recovery samples

....................................................................................9 

### Table 2 

### Concentrations of in 

### test medium (range-finding test)

......... 10 Table 

### 3 Concentrations of in test medium (first limit test)

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

### 11 Table 4 

### Concentrations of in test medium (final test}

........................ 12 FIGURES Figure 1 GC chromatograms ofa 60.0 

### mgll calibration solution [top; res.ld. 3833J and blank M2-medlum Ibottom; res.id. 4031]

..................................... 13 [top; res.id. 35191. 100 mgll [middle; res.id. 3528] and 259 mgll [bottom; res.ld. Figure 2 GC chromatograms of 

### procedural recovery samples at concentrations of 10.1 mg/l 

### 3531]
.....................................................................,................................................. 13 Figure 3 GC chromatograms of blank control samples taken 

### att=O hours ftop; res.ld. 3497]. 

### a1 Figure 4 GC chromatograms of nominal 257 mgn samples taken att=24 hours [top; res.id. t=24 hours [middle: res.id. 4032] and at 

### t=72 hours [bottom: res.id. 4034]

............ 14 4036] and at t=72 hours [bottom; res.id. 4040]

....................................................... 14 Figure 

### 5 GC chromatograms 

### of nominal 257 mg/l without 

### algae samples taken at t=Q 

### hours [top; res.id. 3513]. at t=24 hours [middle; 

### res.ld. 4038] and att=72 hours [bottom; res.id. 4042]

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

### 15

- Page 2­

### 2. REPORT APPROVAL


### PRINCIPAL SCIENTIST: (Analytical Chemistry)



### -Page3·3. INTRODUCTION 

### 3.1. Preface Study plan Start : 30 May 2005 (analytical study) Completion : 20 September2005 

### 3.2. Aim 

### ofthe study The purpose ofthe analytical study was to determine the actual 

### concentrations in samples taken from the test solutions used during the ecotoxlchy test. 

### 4. MATERIALS AND METHODS 

### 4.1. Reagents Methanol HPLC-grade. labscan, DubUn, Ireland MiIIi-Qwater Tap water purified by reversed osmosis and subsequently passed over activated carbon and ionexchange cartridges; Millipore Corp, Bedford, MA, USA ISO-medium Medium formulated using Milli-Ro water (tap water purified 

### by reverse osmosis; Millipore Corp.• Bedford, Mass., USA) with the following composition: CaCI2.2HtO 293.8 mgll MgSO

..7H:!O 123.3 mgll**NaHCO~** 64.8 mg/l KCI 5.8mg11 M2-medium 

### see main report Tapwater 4.2. Sample. 

### All samples not analysed on the sampling day were stored frozen. Stability ofthe samples under these conditions was determined during flDevelopment and validation of an analytical method for the analysis of In MIlIi-Q 

### water". On the day of analysis, 

### the frozen samples

### were defrosted at room temperature. The samples (10 ml) were analysed using headspace (see 4.3.1 analytical method).

-Page4­

### 4.3. Analysis 4.3.1. Analytical method Quantitative analysis was 

### based on the test substance peak in the GC chromatogram of 

### Analyticalconditions Column Slationary phase CP Porabond Q 

### Dimensions 

### 25 

### m x0.25 mm, d,= 

### 3

### j.lm Brand Varian, Mulgrave, VICtoria, Australia Temperature program Initial 

### temperature 40 DC 

### Initial time 

### 1 min Rate 

### 1 20 ·C/mln 

### Temperature 1 

### 190 ·C Rate 

### 2 50 ·C/min 

### Final 

### temperature 250·C Hold time 

### 3.3 min Carrier gas Helium Column flow 1 

### mVmin Injection Splitless Temperature 

### 250 ·C Splitless time 2 minutes Injection volume 

### 500**~I** 

### (Headspace) Injection speed 50 !-II/sec Headspace conditions Sample 

### 10 ml of an aqueous sample in a

### 20 ml 

### vial Incubation temperature 

### 50·C Incubation time 

### 30 minutes Syringe temperature 

### 110·C Detection FlO (250 ·C; Hydrogen: 40 ml/min; Air: 

### 450 mllmin) 

### 4.3.2. Calibration During the range finding test, calibration solutions in ISO-medium were made up from two standard solutions of in methanol. During the first limittest, calibration solutions in M2-medium were made up from two standard • solutions of in methanol. During the final test, calibration solutions in tapwater were made up from two or three standard solutions of in methanol. The differences in matrix between calibration and ecotoxlcity samples was tested and had no influence on the quantilBtive results.



### - Page 5·4.3.3. Injections 

### Calibration solutions were injected in duplicate. Procedural recovery samples and test samples were analysed by single Injection. 

### 4.3.4. Data acquisition and data processing Data acquisition 

### and data processing was perfonned 

### using Empower software (Waters. Milford. • MA. USA)

### version 5.00. 4.3.5. Procedural recovery 

### samples Standard solutions of in 

### methanol and dilutions of these 

### solutions with 

### methanol were used as spiking solutions. During the 

### range-finding test. blank test medium {10**m~** was 

### spiked at concentration levels of 0.101 mgll and 101 

### mgtli using 10.1 mg/l and 10085 mgll spiking solutions in methanol, respectively. During the first limit

### test, blank test medium 

### (10**m~** 

### was spiked at concentration levels 

### of0.101 mgll and 

### 101 mgll' using 10.1 mg/l and 

### 10115 mgtl spiking solutions in 

### methanol. respectively. 

### At 0.101 mgll one and at 101 mg/l five samples were prepared and treated as described in paragraph 4.2 

### 'Samples' and analysed. The recovery was 

### calculated for each sample. 

### During the final 

### test, blank test medium (10 mil

### was spiked at concentration levels of 

### approximately 10 

### mgll. 100 mgll and 260 mgJI using spiking solutions in methanol at concentration levels ofapproximately, 1000 mgll, 10000 mg/l and 26000 mgtl, respectively. 

### 4.4. Interpretation 4.4.1. Calculations All 

### calculations were performed using non-rounded values. Rounded values are reported in the tables. Therefore, some differences might be observed when calculating the parameters 

### as mentioned in the tables. 

### 4.4.2. Formulas Genera! _ 1 n 

### Mean:**x=-~** Xi n 1-1 

### where Xi 

### = measured value 

### n 

### = number of 

### measurements

n L(Xi-x)2 Sn-I= 11..:;;;"'

...__­ 1 

### Standard deviation (n-I) 

### Coefficient ofvariation

, Concentrations were nol conected for spiking volume 

### -page6­Calibration Response: R = Peak area testsubstance [units] Calibration curve: The response was correlated with the test substance concentration, using linear regression analysis (least squares method; weighting factor (1/concentration2)).



### R 

# = response calibration solution [units] 

### C 

# = concentration oftest substance in calibration solution [mgll) 

### a = 

### slope 

### lunits*lImg] b 

### = intercept [units]**~amples** Concentration 

### ofASAHIKLlN AE-3OO0 analysed 

### in the samples: C; (R-b}*d [mg"] 

### a R ;;: response sample [units] d 

### = dilution factor 

### a 

# = slope [units.VmgJ 

### b 

# = intercept 

### [units] Recovery of procedural recovery 

### samples: Concentration analysed

.. 100 

### [%] Concentration prepared Concentration analysed Relative to nominal concentration: Concentration nominal • 100 

### [%]

Concentration analysed at 

### t

## =x hours

.. 100 

### [%] Relative to initial concentration: Concentration analysed 

### at t = 0 hours



### - Page 7·5. RESULTS 5.1. Calibration curves 

### On each day ofanalysis, a calibration curve was constructed using at leastsix concentration levels. 

### At each concentration level, two responses were used. The coefficient of correlation was in excess of0.99 for each calibration curve. Figure 1 shows chromatograms ofa calibration solution and blank methanol. 5.2. Sampies 

### 5.2.1. Procedural recovery samples 

### Figure 

### 2 shows chromatograms of procedural recovery samples. The results for the procedural recovery samples are summarized in Table 

### 1. Mean recoveries 

### ofprocedural recovery samples 

### are required to 

### be in the range 70-110%. (Mean) recoveries ofthe procedural recovery samples were between 86% and 113%. 

### The reason for the relatively high recovery at atest substance level of0.101 mgll determined on 

### 16-06-05 

### was unclear, but the result was considered acceptable. 5.2.2. Testsamplas 

### Figures 3 - 5 show chromatograms 

### ofblank control samples. nominal 257 mgll samples and nominal 257 

### mgl1 without algae samples from the final test The results forthe test samples are summarized in 

### Tables 2 - 4. During the range-finding test, the 

### test substance concentration in the nominal 100 mgll test samples taken at t=24 and nominal 10 mg/l taken att=72 were below the limitofquantification (lOQ) ofthe analytical method. During the first limittest, a small peak atthe retention time ofthe test substance was observed 

# in. the chromatograms ofthe blank

### test samples (nominal 0 mgll). Because a similar peakwas found in blanks of methanol itwas concluded that the compound did not derive from test medium. For the blank test samples, the response ofthe interference was below the limitofquantification (lOO) 

### ofthe 

### analytical method. For the nominal 147 mgll test samples with and without algae, the maximum contribution ofthe compound to the test sample with the smallest peak area was 0.6%, and was therefore not considered to be a Significant interference.

. During the final test. 

## a small peak at

### the retention time ofthe 

### test substance was observed. This peak

### was probably caused by a memory effectofthe analytical system. For the test samples of nominal 257 mg/l samples with and without algae, the maximum contribution ofthe blank peak to the test sample with the smallest peak area was 

### 2.2%, and was therefore notconsidered 

### to be a significant interference.

- Page 8­TABLES Table 1 Procedural recovery samples 

### Date of 

### preparation [dd-mm-yy] 

### Date 

### of analysis [dd-mm-yy] 

### Concentration nominal

[mg/l] 

### Concentration analysed

[mgtn 

### Recovery 

### rib] Mean 

### recovery (Coefficient of

variation) 

### r%1 30-OS-05 30-05-05 0.101 0.103 102 30-05-05 30-05-05 101 76.3 76 101 75.0 74 

### 86 101 101 100 (13) 101 96.1 95 101 87.5 87 16-06-05 16-06-05 0.101 0.115 113 16-06-05 16-06-05 101 102 101 101 99.5 98 100 101 101 100 (1.0) 101 101 

### 99 101 101 100 13-09--05 13-09-05 

### 10.1 11.0 109 107 10.1 10.6 106 (2.0) 10.1 10.6 105 13·09-05 13-09...(15 100 103 103 106 100 108 108 (2·n 

### 100 106 

### 106 13-09-05 13-09-05 259 261 108 259 287 111 108 259 281 108 (1.6) 259 275 106 259 280 108 19-09-05 19-09-05 10.0 9.09 91 89 10.0 

### 9.04 90 (3.2) 10.0 8.57 

### 86 19-09-05 19-09-05 100 78.2 78 76 100 77.7 78 (5.6) 100 70.7 71 19-09-05 19-09-05 260 221 85 82 260 206 79 (3.8) 260 217 83 

# .

- Page 9 ­

### Table 2 Coneentrations 

### of In 

### test medium (range-finding 

### test) Time of sampling



### [hours) Date of sampling Idd-mm-yy] 

### Date of analysis Idd-mm-yy] 1**~.** 

### Nominal

[mgll} Concentration Relative to 

### Analysed nominal

[mgll] [%] 

### Relative 

### to mean initial 

### [0.41 0**23-05~05** 30-05-05 

### 10 1.03 

### 10 10 1.02 

### 10 100 14.3 

### 14 100 14.9 

### 15 

### 24 24-05-05 30-05-05 10 0.132 1 13 10 0.132 1 13 100 2.18 2 15 100 

### <LOa n.a. n.a. 

# . 72 26-05-05 30-05-05 10 

### <Loa 

### n.a. n.a. 10 0.255 3 25 I 100 

## 1.n 2 12 100 1.76 

### 2 12 I Samples were fro;ten until analysis. lOQ LImH of quantification was 0.100 mgn . "Dewlopmenland validation ofan analytical method for the analysis of In MilII..Q water). n.a. Not applicable



### - Page 10­

### Table3 Concentrations of In

### t8St medium (first limittest) 

# I Timeof

. sampling [hours) 

### Date of sampling [dd-mm-yy] Date of 

### analysis [dd-mm-yy] Concentration Nominal

[mgll] Analysed [mgll] Relative to nomina!

[%] 

### Relative to mean initial [%] 

### 0 13-06-05 16-06-05 1 

### 0 <LOO 3 

### 0 <LOO3 

### 147 

### 56.9 39 147 57.9 39 1472 n.i 

### 1472 56.9 

### 39 

### 24 14-06-05 16-06-05 1 

### 0 <LOa**~** 

### 0 <Loa 3 

### 147 6.11 

### 4 11 147 6.47 4 11 1472 6.86 5 12 1472 6.52 4 12 72 16-06-05 16-06-05 

### 0 <Loo3 

### 0 <LOQ' 

### 147 147 2.10 2.04 1 1 4 4 

# . 1472 2.36 2 4 1472 2.29 2 4 Samples were frozen until analy&ls. Wrthoul algae. LOQ' A small peakwas observed at the retention tine oftest substance, but its respOJ'\M

### was not coosldeted to derIVe from test aample since Itwas also observed in blank methanol. Umit ofquantlfteatlon was 0.100 mgll (See "Development and validation ofan analytical method forthe analysis of In MiIIl-Q water"). n.l. NoIlnJected, dueto asystem error.



### - Page 11

­Table 4 Concentrations of in

### test medium (final test) Time of**samp~ng** [hours] Date of sampHng [dd-mm-yy] Date of analysis [dd-mm-yy] Concentration Nominal



### [mg/l] Analysed [mgll] Relative to nominal

[%] Relative to mean initial [%1 0 13-09-05 13-09-05 0 n.d. n.a. 0 n.d. n.a. 257 3 212 82 257 3 214 83 24 14-09-05 19-09-05 1 0 2 n.a. n.a. 0 2 n.a. n.a. 257 12.6 5 6 257 13.3 5 

### 6 257 3 14.6 6 7 257 3 13.4 5 6 72 16-09-05 19-09-05 1 0 2 n.a. n.a. 0 2 n.a. n.a. 257 4.99 

### 2 2 257 7.61 3 

### 4 257 3 6.57 3 

### 3 257 3 

### 5.83 2 

### 3 Samples were frozen until analysis. A smal peak was observed In these nominal 0 mgtl &les. this Wla probably II memory effect ofthe analytical system. The maximum contribution oflhls peak 10 the samples was 2.2% and this was not con.sldered to be a significan1interference. Without algae. n.d. Not detected n.a. Not applicable

- Page 12­FIGURES



# - _



# .. ..



# _

.. ,'


# .....



# _

..

..... 

# '-

....



# ......



# ..

# ­ ,,--



# .­



# -

....

.... 

# ... 

# ... ..

# ... 

# . .... 

# - Figure 1 GC chromatograms ofa 80.0 mgn calibration solution [top: 

### res.id. 3833] and blank M2-medium [bottom; res.ld.4031].

# ,~**,~**

-.. 

### 'I.-"



# _

...



### •

# ... .... ,

# .. ,

# . 

# - .... II." oj• 

# ­ Figure

### 2 GC chromatograms ofprocedural recovery samples at concentrations of 

### 10.1 man [top; 

### res.ld. 3519], 100 mg/l 

### [middle; res.id. 3528] 

### and 259 

### mglJ [bottomj res.ld. 3531].



### - Page 13­

# ....



# .... t 

## "

# ..

....

.. 

# ... 

# ... ,... t



## .....



# ,­

, 

# -

.....

..... t



# .....

.....
.o. 

# -

"

# ... 

# ... 

# J v



# ---
II

# !**~** 

### = Uo 

# .... 

# '.., 'lilt' , 

# ..... T
... • • flO 

# ... 

# ... .... 

# . ... FIgure 3 GC cbromatograms of blankcontrol samplestaken attacO hours [top; res.ld. 349'1]. 

### att=24 hou

... [middle; res.ld. 4032] and 

### at t-72 hours 

### [bottom; res.id.40341.



# _

.. 

# - ,,"''''



# -
"'

# .

....

,

# .....



# .... "


# .....



# - . 

# ­

....

,. ,

# .. ,--'**~.~ .:.~'** **~.** ,-'**~.~'-r-r.:.~~,---.-.~'~' -rl'J.a~'** **~....,.:."--;:;:""""~~"""",~,,,,,** 

### ....**!---o~~~.r·~.** 2
... ... ... •••**~,,,,,,,,,,,,,,,,~.......-l** 

# ...­ Figure4 GC chromatograms of nominal 257 mgll samples taken at 1-24 hours [top; res.ld. 4036] and at t-72 hours [bottomj res.ld. 4040].

- Page 14­

# - _.**-~**



## _ .. -..



### • 

# _. _.

_

.

.......



# .....

'0. 

### ,

# ... 

# ... 

# .. obo 

# . 

# - ... 

# . .. .... ..... 

# .... 

# ..

# .. d. >lot 

# - Figure 5 GC chromatograms of nominal 267 mg/I withoutalgae samplestaken at 

### t=O hours [toPi res.ld. 3513], att-24 hours [middle; res.ld. 4038] and at t-72 hours [bottom: res.ld.4042].

- Page 15­

## P-04-635 REPORT

StudyTitle 

## 96-HOUR ACUTE TOXICITY STUDY IN CARPWITH (FLOW-THROUGH)

Study completion date 28

### October2005



### }i!st FacilHy



### Laboratory PrQject Identification



### . Pagel of 

### 35
.

### 1. CONTENTS 1. CONTENTS

......................'

.....................................................................................................2 2. STATEMENT OF GLP COMPLIANCE

..................................................................................4 3. QUALITY ASSURANCE STATEMENT

.................................................................................5 4. SUMMARy

.............................................................................................................................6 

### 5. INTRODUCTION

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

### 7 5.1. Preface

...........................................................................................................................7 5.2. Aim of 

### the study

..............................................................................................................7 5.3. Guidelines

.......................................................................................................................7 5.4. Storage and retention of records and materials

.............................................................7 5.5. Definitions

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

# a 6. MATERIALS AND METHODS

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

# a 6.1.1. Test substance

.....................................................................................................8 6.1.2. Reference substance

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

### 9 6.1.3. Preliminary data

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

### 9 6.2. Test System

....................................................................................................................9 

### 6.3. Holding

.......................................................:...................................................................

### 9 6.4. Range-finding 

### test (static)

............................................................................................ 10 

### 6.5. Rrst and second limit flow-through test
........................................................................ 

### 10 6.5.1. Conoentrations

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

### 10 6.52. Dosing system
.................................................................................................... 

### 10 6.5.3. Test procedure and conditions

........................................................................... 12 6.5.4. Sampling for analysis of test concentrations

..................................................... 12 6.5.5. Measurements and recordings

.......................................................................... 12 

### 6.6. Interpretation

......,.........................................................................................................

### 13 6.6.1. Data handling

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

### 13 6.62. Acceptability of the test
......................................................................................

### 13 6.7. List of protocol deviations

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

### 13 7. RESULTS

............................................................................................................................. 14 

### 7.1. Static range-finding test
................................................................................................ 14 7.2. Firstflow-through test
................................................................................................... 14 7.2.1. Measured ooncentrations

................................................................................... 14 7.22. Mortality and other effects

..................................................................................15 7.2.3. Experimental conditions

..................................................................................... 15 7.2.4. Discussion on results 1st limit tesL

..................................................................... 15 

### 7.3. Second flow-through te8t.

............................................................................................. 15 

### 7.3.1. Measured concentration8

................................................................................... 15 

### 7.3.2. Mortality and other effects

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

### '0. 7.3.3. Experimental conditions

..................................................................................... 16 

### 7.3.4. Determination 

### of effectconcentrations

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

### 16 

### 8. DISCUSSION

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

### '7 9. CONCLUSION

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

### '7 TABLES Table 1 DOSing program 181 limittest
.............................................................................. 11 Table 

### 2 Dosing program limit test
.............................................................................. 11 Table 

### 3 Incidence of mortalityand total mortality during the range-finding test
............. 14 Table4 Mean measured collC9ntrations versus nominal concentration (1 S1 limittest)

... 14 TableS Incidence of mortality and total mortality during the 1st limittest
....................... 15 Table 6 Mean measured concentrations versus nominal concentration 

### (2'" limit test)

.................................................................................................................... 16 Table 7 Incidenceof mortality and total mortality during the 2nd limittest
...................... , 6 Table 

### 8 Effect parameters
............................................................................................... 16 Table 

### 9 pH-values and ,dissolved oxygen ooncentrations (mgll) during the 111 limit te6t.

..................................................................................................................... 18 Table 10 Temperatures (OC) measured during the 1" limit test
........................................ 18 Table 11 pH-values and dissolved oxygen concentrations (mgll) during the 2nd test
... .... 18 Table 12 Temperatures 

### (OC) measured during the 2nd test
............................................... 18



### - Page 2·FIGURES Figure 

### 1 Flow-through system, zt4l1mlt

### test
..................................................................... 11 APPENDICES APPENDIX I TEST CONDITIONS

.......................................................................................... 18 APPENDIX II REFERENCE TEST APPENDIX IV ANALYTICAL REPORT ........................................................................................... 19 APPENDIX III ANALYSES OF TAP WATER (Not performed underGLP)

...............................20

·Page3­

### 2. STATEMENT OF GLP COMPLIANCE

The study described in this report has 

### been correctly reported 

### and was conducted In compliance with: 

### The Organization for Economic Cooperation and Development (OECD) Good Laboratory Practice Guidelines (1997). 

### Which essentiallyconform to: The 

### United States Food and Drug Administration Good Laboratory Practice Regulations. The 

### United States Environmental Protection Agency Good Laboratory Practice Regulations. The sponsor is responsible for Good laboratory Practice (GlP) 

### compliance for 

### all test substance Information unless determIned by 

### Tap water analyses were 

### not performed under GlP

### and therefore excluded from this GLP 

### statement.

Study Director Head of In Vitro & 

### Environmental Toxicology

### ~Dat9:**.~s..I** 

# ..**Od-.Q~.e..f** 

### ..**~.s:-** 

# .......... Date:

.......

# ~¢'.ap..Q~............... 

# ... .

·Page4·3. QUALITY ASSURANCE STATEMENT

This report 

### was inspected by the Quality Assurance Unit to confirm thatthe methods 

### and results accurately 

### and completely reflect the raw data. The 

### dates 

### ofQuality Assurance 

### inspections are 

### given below. During the on-site process Inspections procedures applicable tothis type of studywere 

### Inspected. Type of 

### Inspections Study

Process PhaseJProcess
Protocol Ameminent 

### 1 of protocol Ameminent

### 2 of protocol Amendment 3 of protocol Amendment 4 

### of protocol Amendment 

### 5 of protocol Amendment 

### 6 of protocol Amendment

### 7 of protocol Amendment

# aof protocol Report Environmentaltoxicology 

### Test substance handling Exposure ObservationsIMeasurements Analytical and physical chemistry Test

### substanqe handling Observations/Measurements 

### Start Inspection date 

### Q3-Sep-04 Oa-May-05 22-Jun-05 30.Jun-05 22-Jut-os 08-Sep-OS 3O-Sep-OS 07-Oct-OS 

### 2a-Oct-OS 28-Oct-oS 08-Aug-OS

20.Jun-05 2D-Sep-05 

## End Inspection date 

### 03-Sep-04 03-May-05 22-Jun-05 3Q-Jun-05 22-Jul-05 OS-Sep-oS 3O-Sep-05 07-0ot-OS 28-0c1-DS 28-0ot-05 19-Aug-05

29.Jun-05 26-Sep-OS Reporting· date 03-Sep-04 

### 03-May-05 22-Jun-oS 30-Jun-05 22.Jul-05 OS-Sep-OS 3O-Sep-05 07-00t-05 

### 28-0c!-OS 2a.oct-05 19-Aug-05

05.JuJ-05 Z7-Sep-OS

Head of 

### Quality Assurance



### -Pages­4. SUMMARY 96-Hour Acute Toxicity Study in Carp with The study procedure described in this report was based on the ISO International Standard 

### 7346:3: Flow-1hrough method. 1996. In addition. the procedures were designed to meet the test methods and validity cnterla of the EEC directive 92/69, Part C.1, 1992 and the DECO guideline No. 203, 1992. The batch 

### of tested was aclear and colourless Ilqu1d with a purity 

### of 99.985%. was completely soluble in test medium at the concentrations tested and known to be extremely volatile. The project started with a static range-finding test exposing three carp per concentratIon to nominal •concentrations of 0.1, 1.0, 10and 100 mgll. No mortality or clinIcal effects were observed at any of

### the

### test concentrations during the test 

### period. Analytical results of samples 

### taken from 

### 100 mgllshowed that the mean measured concentrations were

. approximately 5 mg/l during the first 24 hours 

### of exposure and 1 mgll at the end of the test period.

Based on these low recoveries It was decidedto continue testing applying a flow-through system. A limittest was performed in an airtight closed flow-through system with continuous renewal of test solutions. A volume of 150,pl pure test substance was closed every 

### 5 minutes and mixed with tap water at aflow rate of 12 LIh. This flow resulted in 8 complete exchanges per day. Seven fish per concentration were exposed to a blank..control and a nominal concentration of 221 

### mg/1 for 

## a maximum of 96 hours. Analyses of samples taken one day before the start of

### the

### test showed a mean measured concentration of 84 mg/l, indicating that the system functioned sufficiently 

### in reaching an actual concentration closeto the target of 100 mgll. The mean measured exposure concentration during the 96-hour test period corresponded to 73 mgll. As this concentration 

### was more than 20% below the regulatory limit concentration of 100 mg/l and fish were significantly affected In their swimming behaviour at a mean concentration of 73 mgll, it was decided to continue with an additional limit test with improved mixing and increased renewal rate. The second limit test 

### was also perfonned in an airtight closed flow-through system with continuous renewal of test solutions. Avolume of 

### 176 pi pure test substance was dosed every 5 minutes applying two diluters and mixed with tap water at an increased flow rate of 14 LJh and smallertest vessels. This resulted in 34 complete exchanges per day. Seven fish per concentration were exposed to a blank..control and a nominal concentration of 221 mg/l for a maximum of 96 hours. Two fish died during the test period, while the surviving fish were discoloured and showed loss of righting reflex. The mean measured concentration 

### was 76 mgll, which was only slightly higher than the mean concentration measured in the first study despite the improvements made to thetest system. Other attempts were made to reach a higher exposure concentration**(~1** 00 mg/l) in test medium. ThIs Included a further increase of the flow rate, change of the nominal concentrations and even included extra mixing vessels. All 

### ofthese did not result 

### in measured concentrations that were higherthan the concentratIons measured in the first two limit studies. The maximum achieved concentration again approximated 70 mg/l. On average. a recovery of30% 

### was found indicating a loss of 70%. In conclusion: with the present extremely volalDe substance it was not possible to test flsh In a stable flow-through test system at a concentration 

### ofat least 100 mg/l. The study met the acceptabilitycriteria prescribed by the protocol and was considered valid. Under the flow-through conditions of the presenl test Induced less than 50% mortality In carp at a mean measured test concentration of 76 mg/l, the highest concentration that could be achieved and maintained stable In tap water. Based on the observations of preCipitate in the form of droplets in both limit tests, it cannot be excluded that fish 

### were, at leastoccaSionally. exposed 

### to pure lest substance. In conclusion, the 96h-LCso exceeded a mean measured concentration of 

### 76 mg/l. -Page6­5. INTRODUCTION



### .5.1
. Preface Sponsor
Study Monitor Test Facility

Study Director Technical Coordinator Principal Sclentlst Study Plan Start exposure 

### 23 May2005 Completion exposure 17 September 2005 Completion of

### the project 05 October 2005 5.2. Aim ofthe study The purpose of the studywas to evaluate the test substance for Its abilityto generate acute toxic effects in Cyprinus carpio during an exposure period of96 hours and, if posSible, to determine the LCso at ali observation times. 5.3. GuIdelines The study procedure described in this report 

### was based on the ISO International Standard 7346-3: 'Water quality
- Determination of the acute lethal toxicityof substances to a freshwater fish [Brachydsnfo ferlo Hamilton-Buchanan (Teleostel, Cyprlnldae)r
- Part 3: Flow-through method, Second edition, 1996-06-15. In addition, the procedure is designed to meet the test methods and validity criteria prescribed by the following guidelines: • European Economic Community (EEC), EEC directive 92169. Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.1. ·Acute toxicity • forfish". • The OECD guidelines forTesting of Chemicals, guideline No. 203: 

### "Rsh Acute ToxicHy Test", Adopted 

### 17July, 1992. 5.4. Storage and retemlon of records and materials Records and materials pertaining to the study including protocol. raw data, specimens (except specimens requiring refrigeration orfreezing) andthe final report are retained in the archives for a period of at least 10years after finalization of the report. After this period, the sponsor will be contacted to determine whether raw data and specimens should be returned to them, retained or destroyed on their behaH. Those specimens requiring refrigeration orfreezing will be retained by for as long as the quality of the specimens permits evaluation but no longer than three months afterfinalization ofthe report.

- Page 7 ­will retain a test substance sample until the expiry date, but no longerthan 10 years afterfinalization of the report. After this period the sample will bedestroyed. 

### 5.5. Definitions· Fish were considered to be 

### dead when 

### no reaction 

## was obserVed after touching the caudal peduncle and visible breathing movements were absent. In acklition. fish that were convulsing or showing other severe forms 

### of distress not considered transient in nature and likelyto become more severe before the exposure is terminated. will be sacrificed for humane reasons. These fish will be treated as having died In the test. based on OECD guldellne on humane endpoint (ENVlJMIMONO/(2000)7). The 

### LCso 

### is the concentration killing 

### 50% ofthe fish after a 

### defined 

### period of exposure. A Flow-through system 

### Is a system where testsubstance is dosed 

### at fixed time points and mixed with a continuous flow of dilution water to provide continuous renewal ofthe test solutions. 6. MATERIALSAND METHODS 

### 6.1.1. Test subslance Limit test 1 and 2 

### with fish; Identification Structure
Molecularformula Molecularweight CAS Number Descriptlon Clear colourless liquid Batch Purity 99.985% 

### Test substance storage In refrigerator In the dark Stability under storage conditions Stable expirydate 28 February 2006 Specific Gravity 1.47 Stability in water
. Not indicated Extra attempts to reach higher exposure concentrations following 

### the limittes1s with fish: Identification Structure

Molecular formula Molecularweight CAS Number 

### Description Clear colourless liquid Batch Purity 99.989% 

### Test substance storage In refrigerator (2-8·C) 

### In the dark Stability under storage conditions Not indicated Expiry date 26 September 2006 (allocated by 1 year after • 

### recerpt of the test substance)

- PageS­

### 6.1.2. Reference substance This report includesthe results'of a referen<:e 

### testwith pentachlorophenol (PCP) (Appendix I). 

### 6.1.3. Preliminarydata The water solubility of at20.1 ± 

### OSC is 

### 0.805 gil using the flask method 

### Further essential information obtained from physlco-chemical studies performed at were 

### the vapour pressure being 31 kPa at 25"C, freezing point <-81 "C, boiling pOint 

### 56"Cand a log**~of** 

### 2.54. The Log 

### Pow of 

### was 2.18 according to infonnation supplied 

### by the sponsor. From these data. 

### it can be concluded that the test substance will evaporate rapidly at room temperature as pure substance but also from aQueous solutions (calculated Henrycoefficient is 7.24.1as Fa m3 mor'). concentrations showed to be stable during freezing and thawing of samples 

### In a deep-freeze 6.2. Test System Species Carp 

### (Cyprfnus carpio, Teleostel. Cyprinidae) Linnaeus, 1758. Source


### Mean length 1 Range.finding test: 2.3 

### ± 0.1 cm 

### First limit test 

### 2.6 ±

### 0.2 cm Second 

### limit 

### test: 2.2 ± 0.1 

### em Mean weight' Range-finding test: 

### 0.33 ± 

### 0.08 9 First limit

### test: 

### 0.56 :t:

### 0.14 9 Second limit 

### test: 0.29 ± 0.08 9 Characteristics F1 from a

### single parent-pair bred in UV-treated water. Reason for selection This system has been 

### selected as an internationally accepted species. Total fish used 

### 40 

### 6.3. Holding Quarantine! Aoclimatisation At least 12 days aftar delivery. Medium 

### ISO·medium, formulated using 

### Mllli·RO water 

### (tapwater purified 

### by reverse osmosis; Mlllipore Corp., Bedford, Mass., USA) 

### with the following composition: C8C1;.2H;O 293.8 

### mWI MgSO •.7HzO 123.3 

### IYI9'I NaHCo. 64.8 

### mo'I KCI 5.8 mgII 

### Measurements pH, nitrate 

### and nitrite concentraUon 

### and ammonia concentration: once a week. 

### Temperature: avery 

### day. Feeding Daily with Nutra orArtemia.

1 Ten fish ofthe batch used for Ihe lest, were weighed and measured priorto the start ofthetest.

- Page 9­Acclimatisation Following a48-hour settling-in period the fish were allowed to acclimatisetothe test medium (tap-water) wfthout

### test substance for at least seven days. Validity of batch In the batch of fish used for the 

### test, mortality during the seven days priorto the start of 

### the test was less than 

### 5%. 

### 6.4. Range-finding test 

### (staUc) A static range-finding test was performed 

### to provide infoll11ation about the range of oonoentrati0n8 to be used In the final 

### test. Test procedure and conditions were similar

### to those applied in the limit testa wHh the following exceptions: a static test system 

### was used in 

### which three 

### carp

### per concentration 

### were exposed to nominal concentrations of 

### 0.1 

### to 100 mgllincreasing by a facfOr 

### of 10. Dissolved oxygen concentrations, temperature and pH were only measured in the lowest and highest test concentration. Duplicate samples for possible analyses Were taken from all test solutions at the start, after 

### 24 and 

### 96 hours of exposure. Preparation forthe range-finding test started with 

### tast solutions prepared at 10and 1 00 mg/l applying 

### 1 and 

### 13 minutes 

### of magnetic stirring. respectively. Care was taken that during preparation 

### as little 

### as possible volatilisation 

### occurred (weighing and preparation in 

### clOsed vessels which alreadycontained a sman volume oftest medium. short preparation period). The lower

### testconcentrations were prepared bydilution of the test solution of 10 mgll. The final test • solu1ions were all clear and colourless. Part of the test solutions was used forthe simultaneously performed acute toxicity test with Daphnia magna 8.5. First and second limitflow-through 

### test 6.5.1. Concentrations Nominal 

### 221 mg/!, expected to coJTespond to

## an actual concentration 

### close to 100 mgn. Controls Test medium withouttest substance or other additives (blank-control). 6.5.2. Dosing system 

### First limittest; Approximately 75 ml 

### 01 pure test substanoe 

### was collected daily in an airtight sealed vial and used as 'stock' solution for dosing. The pure test substance 

### was dosed via a computercontrolled system in combination with a dispenser (Gnson). 

### VIa this system the dosed volumes entered a mixingflask separatelyfrom the 

### tapwatersupply. The tap water was supplied via a flow meterwith a flow rate of 

### ca. 121111. In the mixing flask the dosed volumes and the tap water 

### were mixed under continuous stirrinQ. 

### Thetest solution coming from the mixing flask entered • first avessel containing Daphnia 

### andthen a vessel containing the fish. The whole system was checked daily. The dosing program Is described In Table 1. The dosed volume of 

### 1.8 ml per hour in combination 

### with a tap-water flow of 

### 12 I!tres per hour results in a nominal concentration of 

### 221 mg/!. However, considering 1he rapid decrease of concentration in water due to the high volatility and the 

### results obtained 

### In a previously performed study with algae 

### itwas expected 

### thatthis dosi1g regime would give an actual concentration close 

### to 100mgll.

-Page 10­

### Table 1 

### Dosing program 1 st 

### limit test Stock solution Dosingvolumes stock (0.5 ml syringe) Tapwater Nominal concentration Expeo1ed concentration Per cycle pL/Smin mUhour Llhour (mgIL) (mglL) Pure 

### 150 1.8 12 

### 221 100 

### §econd limit testj 

### Approximately 70 ml of

### pure 

### test substance was collecred daily and dMded over two 

### airtight sealed vials and used 

### as 'stock' solution for dosing. 

### The pure test substance 

### was dosed via a computer-controlled system in combination with two 

### dispensers (Gilson). Via this system the dosed volumes entered one mixing flask separatelyfrom the tap watersupply. The tap water 

### (14 I/h) 

### was supplied via twoflow meters, each 

### with aflow rate ofca. 

### 7 1Ih. In the mixing flask 

### the dosed volumes 

### and the

### tap water 

### were mixed undercontinuous stirring. The whole system 

## was checked dally. 

### The dosing program Is 

### described In Table 2 

### and a diagram 

### of the system is presented 

### in FlQure 

### 1. Tabfe2 

### Dosing program 2nd 

### limittest stock solution

Pure Pure Dosing volumes stock (0.5 ml syringe) Percycl8 

### , pL/5 min mUhour 88 88



### Mixing Flow Yassel 

### meter1.056 1.058 Tap 

### water
Llhour

7 7 Nomina. Expected concentration concentraUon (mgIL) (mgIL) 221 100



### Tes! vessel alrtlghC closed 

### Figure 1 Flow-through system, 2nd 

### limittest

- Page 

### 11 •8.s.3. Test procedure and conditions Test duration 96 hours Test type Flow-through, continuous renewal of test media. Test vessels 181 limit test: 35 litres; 2nd limittest: 10 Iltres. Both consisting of glass plates sealed with 

### a thin fjlm of silicone and covered airtight by a removable Perspex plate. Exchange rates 

### 1at limittest: 

### 8 times per day. 2nd limit test 34 times per day. Test medium Tap water (data of analysis are attached). Number of fish 

### 7 fish perconcentration and control. Loading 

### 1 81 limit test 

### ca. 0.014

### g fishllitre/day. 

### 2nd limittest: 

### ca. 0.006 9flsMltreiday. Illumination 

### 16hours photoperiod daily. Aeration The 

### test media were not aerated during the test. Feeding No feeding from 

### 48 hours prior to the test and during the tolal test period. Introduction of fish 1 day

## (2"" limit) or 2 days (1 81 limit) after the stan of the dOSing system to allow stabilisation of the test concentrations. Euthanasia At the end ofthe test the surviving fish were rapidly killed by exposing them to 

### ca. 1.2% ethylene glycol monophenylether in water. 

### 6.5.4. Sampling for

### Malysis oftest 

### concen'b'atlons During the limittests duplicate samples for analysis were taken according to the schedule below. The method ofanalysis is described in the appended Analytical Aeport (Appendix IV). Sampling: Frequency 

### 181 limit test: ca. 

### 26 hours following the start of dosing, samples were taken from the 221 mgll-limit concentration to check the actual exposure concentration before the start of

### exposure. 1 st and 2nd limit test: at the start, after 48 hours and at the end of the 96-hourtest period samples were taken from the blank-control and 221 mgll. Volume 10 ml from the approximate centre ofthe test vessel. Storage Samples taken after 48 and 

### 96 hours ofexposure were stored In 

### a deep-freeze. 6.5.5. Measurements and recordings Mortality and other effects At 2y'!-3, 24, 48, 72 and 96 hours following the start of exposure and every momlng from day 1to observed for any dead fish.

- Page 12­Dissolved oxygen content, Daily in all vessels, beginning at the start of the test pH and temperature (day 0). 

### 8.6. Interpretation 6.6.1. Data 

### handling Calculation 

### 01 LCeo: No 

### LCso could be calculated because the test substance provedto be non-toxic 

### (lCso > maximum concentration tested). Defining exposure concentrations: The 

### results were based on the mean 

### 01 the concentrations measured in samples taken at the 

### V81lous tIme Intervals. 

### 8.6.2. Acceptability of

### the

### test 1. No mortality orabnonnal behaving fish were observed in thecontrol group. 

### 2. The oxygen concentration W!1S maintained at at least 60%

### 01 the air saturation value throughout the test 

### (> 5 mgll 

### at 22°C). Othertest conditions (PH and temperature) were maintained within the limits prescribed bythe guidelines. 3. The analytical program provtded evidence that the actual test concentrations were generally maintained within ;l; 20% during the test period. 4. The 96h-LCso of the reference chemical for .the stock of fish was in agreement with results obtained previously in our laboratory. 6.7. 

### Ustofprotocol devlatlorts There were no deviations from the protocol.

- Page 13·

### 7. RESULTS 7.1. Static range-finding

### test The results ofthe range-finding test are presented in Table 3. No mortality orclinical effects were observed at any ofthe t. concentrations during the test period. Based on these results samples taken from nominal 

### 10 and 

### 100 mgll 

### were analysed. Analytical results of samples taken from 100 mg/Ishowed thatthe mean maasured concentrations were approximately 5 mgll during the first 24 hours of exposure and 1 mgJI at the end of the test period. The same low percentages of recovery were observed in samples taken from 10 mgll (see Table 2 of the appended Analytical report). Hence, with the 

### test procedure applied actual exposure concentrations were 

### far below nominal and they decreased significantly during 

### the test period. To enable testing actual concentrations close to nominal and to maintain these 

### stable 

### H was decided to continue testing applying a flow-through system. All test conditions were maintained within the limits prescribed bythe protocol. Table 

### 3 Incidence ofmortalityand total mortality duringthe range-flndlngtest 

### Concentration Initial Cumulative mortality Total Mor1allty (%) Nominal

## <rnsan) number 

### offish 3,.h 24h 48h 72h 9611 0.1 3 0 0 0 0 0 0 1.0 3 0 

### 0 0 0 0 0 10 (0.2-<0.1) 3 0 0 0 0 0 0 100 (5 ·1) 3 0 0 0 0 0 0 BelWeen brack&ts: I'Bnge ofactual m&l!IsunKi concentrations


### 7.2. Firstflow-through

### test 7.2.1. Measured concentratIOns The results ofanalysis 

### ofthe samples taken during the first limit test are described In Table 3 

### of the appended Analytical Report. Analyses of the samples taken one day before the start ofthetest showed a mean measured concentration 

### of 84mgll, which Indicated that the system functioned as expected and that the actual concentration was close to the target of 100 mgll. The measured exposure concentrations in each 

### ofthe duplicate samples taken at the various sampling times are shown in Table 4. The difference In duplicate values might be explained by variations In amounts 

### of loss during sampling and the limited solubility in tap water. All results were 

### used to determine the mean exposure concentration during the 96-hourtest period, 

### which correspondedto 73

### mgII. Table4 Mean measured concentrations versus nominal concentration (1at limit 

### test) NomInal concentration Measured Measured t=48h Measured t=96h Mean measured t=Oh

- Page 14­7.2.2. Mortality and othereffects Table 5 shows the mortality data recorded during the first flow-through test. No mortality

### was observed atthe limit concentration tested. However. fish were affected in their swimming behaviour and appearance. Fish were observed to be discoloured during the entire 

### test period. Further. all fish were observed swimming disorientated 

### from 24 hours following exposure and droplets 

### oftest substance were observed on the bottom of the test tank. 

### At the end of

### the test period only one fish 

### was observed swimming disorientated. Hence. this effect diminished during the course of the test. TableS Incidence of mortality andtotal mortalityduring

### the 1- limn

### test COncentration Cumull1tlve mortality 

### 24h 72h 

### 96h



## o o 

## o o o 

## o 7.2.3. experimental conditions The results of measurement of pH. oxygen concentrations and temperature are presented in Appendix 

### I. All test conditions remained within the ranges prescribed bythe protocol (pH: 6.0­ 

### B.S. constant within 

### 1 unit; temperature 

### 20-24"C, constant within 

### 2°C; oxygen> 

### 60%

### of air saturation value). 7.2.4. Discussion on results,- limittest Based on the results obtained In the first limit test It was decided tocontinue with the 

### project. as the mean measured concentration 

### of73 mgll 

### was more than 

### 20% belowthe regulatory limit concentration of 

### 100 

### mgII. This decision 

### was strengthened by the fact that significant effects 

### on swimming behaviour

### were observed at 73 mgll and the water solubilitywas expected to exceed 100mg./!. The rate of renewal oftest solutions 

### was Increased by a factor of four in the second test by decreasing thevolume ofthe test tank 

### from 35to 

### 10 Iltres and increasing the tap water flow rate from 12to 14 Llhour. Hence, test solutions were renewed within one hour.


### 7.3. Second flow-through

### test 7.3.1. Measured concentrations The results of analysis of the samples taken during the second limittest are described in Table 

### 4 ofthe appended Analytical Report. Analyses ofthe samples taken one hourbefore introduction ofthe 

### carp showed 

### a mean measured concentration 

### of86 mg/l, which indicated thatthe system functioned as expected and that the actual concentration was within 

### 20% ofthe target concentration of 

### 100 mg./!. The measured exposure concentrations in each of the duplicate samples taken at thevarious sampling times are shown in Table 

### 6. Again there were significant differences In dupUcate values. The difference In duplicate values might be explained by variations In amounts of loss during sampling, the limited solubillty In tap water and In this case especially the variation in recovery of the analytical method on the day of measurement (see Table 1 of the appended analytical report). All T9SUnS 

### were used to determine the mean exposure concentration during the 96-hour test period. which corresponded to 

### 76 mgll.

- Page 15­TableS Mean measured concentrations versus nominal concentration (2nd limit test) Nominal ooncentratfon MeasuNd t=-1h 60.6/72.5 Measured t:=96 h

82.9/69.1 Mean meuunKI

7.3.2. Mortalityand other effecta Table 7 shows the mortality data recorded during thesecond limit test. Two fish died during the course 

### ofthestudy. The surviving fish were observed to be discoloured during the entire test period. Further, all fish were observed swimming highlydisorientated from 24 hours following exposure as was observed in the 1It limit test. However, this effect did not diminish In time as was observed during 

### the 1 lit limit test. Again precipitation in 

### theform 

### of droplets was observed during the coorse ofexposure. Table 7 Incidence of mortalityandtotal mortalityduringthe2nd IImHtest Concentration Initial Cumulative mortality Total (mgll) number offish 2Y.zh 24h 48h 72h 96h Mortality ("') Blank-control 7 0 0 0 0 0 0 

### 76 7 0 1 1 1 2 29 

### 7.3.3. experimental conditions The results 

### of measurement of 

### pH. oxygen concentrations and temperature are 

### presented In Appendix 

### I. All 

### testconditions remained within 

### the ranges prescribed 

### by the protocol 

### (PH: B.OS.5, constantwithin 1 unit; temperature 20-24"C. constant within 2"0: oxygen> 60% of 

### air saturation value). 7.3.4. Determination of effectconcentrations Table 8 shows the effect parameters basad on the results 

### of both limit studies. The parameters • are based on the mean measured concentration. Table 

### 8 Errect parameters 

# I ParanMlter Concentration (mgll) NOEC <73 24148f12196trLCso >76

- Page 16­

### 8. DISCUSSION Two limittests were performed 

### with the intention of testing an actual exposure concentration of 100mg/l. the regulatory limitconcentration. The mean measured concentration 

### was however 73 and 78 mgll, forthe 11tand the 2nd limittest respectively. This despite

### thefactthat a nominal concentration of 221 mgll was tested and thattest solutions were refreshed >30times per day In the tesl Fish were clearly affected in their swimming behaviour in 

### both tests and two fish died in the 2nd limit. Hence, 

### It could not

### be guaranteed that the LC50was above 100 mgll. Therafore, various attempts with different procedures were tested to reach a higher exposure concentration (lt100 

### mgJ1) in test medium. The

### test system of the 2nd limit

### test applying 10-litre 

### vessels was used.

### Theflow-rate 

## was increased 

### to a maximum of.20 liters perhourto check whether an even higher refreshment rate (48 times/day) resulted in testing higher exposure concentrations. Further,**nomin~** concentrations 

### were Initially lowered to 176 and finally increased to 265 mgll

### to checkwhether any**d~solutlon** problems occurred at higher concentrations. AnaJly, the use of double mixing vessels (in series) to improve dissolution was tested. Dissolution appeared to

### be a problem as precipitate in the form ofdroplets 

### was observed during the course ofthe two limitstudies. In additIon to thls possible problem, sampling at various places In the testvessel was performed to checkthe homogeneity of the test solution. The results of this check showed rather large variations in test concentrations, which strengthens the anticipated dissolution problems, which are likelyto be related to the relatively short mixing time which is again a consequence ofthe hIgh renewal rate. Analytical resuHs showed thatthe measured concentration remained clearly below 70 mgll when testing a lower nominal concentration of 176 mgll (221 mglllnthe lImit 

### tests). This did thus not Improvethe results. At nominally265 mgll, measured concentrations averaged those found in the

### two previous limittests. Hence, even atthis higher concentration a measured ooncentration of 100 mgll could not be reached. Finally, the use of double mixing vessels (In series) to improve dissolution was tested at a nominal concentratIon of 265 mgll. No precipitate in the test vessels 

### was formed when applying

.these exira mixing steps, 

### but analytical results stili remained at the same level as

## was recorded

during the two limittests. Hence, all of these attempts did not result in measured concentrations Ihatwere higherthan 

### the concentrations measured in the first two limit studies. The maxImum achIeved concentration approximated 70 mgll (see Table 5of the appended Analytical report). On average, a recovery of30% 

### was found indicating a loss of 70%. In conclusion: 

### with the present extremelyvolatlle substance 

### It 

### was not possible 

### to test fish in a stable flow-through test system at a concentration of at least 100 mgll.


### 9. CONCLUSION Underthe**flow~through** conditions of the presenttest induced less than 50% mortality in carp at a mean measured test concentration of76 mg/l, the highest exposure concentration that could be achieved and maintained stable in tap water. Based on the observations of precipitate in the form of droplets in both limittests, it cannot be excluded that fish were, at least occasionally, exposed to pure test substance. In conclusion, the 96h-LCM exceeded a mean measured concentration of 76 mg/l.

- Page 17­APPENDIX I TEST CONDmONS


### Elrst 

### limit

### test Table

### 9 pH-yalues and dissolved oxygen concentrations (mg/f) duringthe 111t limit 

### test Concentnltlon 

### Day2 

### Day 3 

### Day4 pH 

### Oz pH 

### Oz pH 

### O:t 8.0 8.6 7.9 8.4 8.0 8.5 8.0 8.6 7.9 8.2 7.9 8.3 Temperatures 

### (OC) measured during

### the 

### 1 lit 

### limit

### test Table 

### 10

Concentration (mg/'l) DIy0 

### Dey 1 

### DIY2 

### Day 3 

### Day4
Blank-oonb'Ol 21.2 21.1 21.0 21.4 21.1 73 21.3 21.3 21.3 21.4 21.4


### Second limit

### test: Table 11 ptf,.yalu•• and dls.olved oxygen concentrations (mglJ) duringthe2nd test

Concentration 

### Day0 

### Day 1 

### Day 2 DayS Day4 (mgll) pH 

### O:t pH 

### O:t pH 02 pH 

### O:t pH 02 Blank-control 7.8 8.9 7.7 9.1 7.8 9.0 7.9 9.0 8.0 9.4 75 7.1 8.7 7.7 

### B.S 7.8 10.1 7.8 8.9 7.9 9.1 Table 12 Temperatures (OC) measured duringthe2nd test

COncentration (mg/'l) Day0 Day1 DIy2 DayS Day4

Blank-control 21.4 21.6 21.3 21.3 21.4 76 21.9 21.9 21.9 21.9 21.S



### -Page1S­APPENDIX II REFERENCETEST 96-hour acute toxicity study in the carp 

### with PCP; The study procedures described In this report were based on the EEC directive 92169, Part C.1, 1992 "Acutetoxicity for·flsh·; and the OECO guideline No. 203: ·Flsh Acute Toxicity Test·, Adopted 17 July, 1992. Start: 

### 17January

### 2005 End: 

### 21 January

### 2005 This referenoe testwas carried O'-lt to check the sensitivityofthe test system as used by The reference substance 

### was pentachlorophenol (PCP. SIGMA, Art. P9441 , Batch 

### 103H3488). Concentrations: 0.10, 0.22 

### and0.46 mgllin ISO-medium. (ncidence ofmortality observed in the reference study:
ConcentraOon

PCP (mg/!) Nominal

0.10 0.22 0.46 Inl1lll1 72h 9th 0 0 5 5 5 5 Tobll Morbility 

### number offish

5 0 5 100 S 100 During the 

### testthe pH, oxygen concentration and the temperature of

### the medium were within the opHmal ranges for fish. Underthe conditions ofthe present test pcp induced 

### no lethal effects in carp

### at 0.10 mgll. The 96h-LC&o for carp exposed to pcpwas 0.15 mg.ll (a 95% confidence interval between 0.10 and 0..22 mgJl) and

### was already reached within 24 hours of exposure. The 96h-LCso for carp is generally between 0.10 and 0.46 mgll 

### based on historical data ofreference tests performed approximatelyevery 3 monthsfrom AprR 1988 untilthe end 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensiHvlties generally observed during the past years. The raw data and reportfrom this 

### studyare kept In 

### the archives. Thetest descrIbed abovewas performed under GLP-conditions with aQA·check.

·Page19­APPENDIX III ANALYSES OFTAP WATER (Not performed underGLP) Date of sampling : 08-06-05 Sample numbers : 2106017 (kamer 25) Laboratory ANALYSIS: ICOMPONENT: Metals: Selenium Arsenic C8dnium Chromium Copper Mercury Lead Zinc Manganese Calcium Magnesium Iron EOX (extractableorganlohalogens) Polycyclic Aromatic Hydrocarbons: Naphtalene Acenaphtytene Acenaphtene Fluorene Phenanthrene Anthracene F1uoranthene Pyrena Benzo(a)antracene Chrysene Benzo(b)fluoranltlene Benzo(k)ftuoranthene Benzo(a)pyrene Dlbenzo(ah)antracene Benzo(ghl)perylene Indeno(123cd)pyrene
PAC'sTotal EPA(16) PAC'sToeal VROM (10) <0.90 <5.0 <0.40 <1.0 29 <0.050 <5.0 19 <0.010 53

6.2 <0.050 <1.0
<0.010 <0.050 <0.010 <0.010 <0.010 <0.0050 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 Ilg/l 1Ig/1 1Ig/1 Ilgll 11911ji) pgll !JgII jJgII mgll mgllll) mgllll) mgll ",gil pgII pgII pgII**~** 

### JIW'I IJg/I IJg/I IJg/I**~** IJg/I JJgJ\ 1Ig11 JJgJ\ 1Ig/1 IJg/I IIg/I

Total aerobic germ-count (22"C) 2 CFU/ml'" Total aerobio germ-counl (37"C) 2 CFU/m1 11 Hardness: 1.6 mmolllji) Nitrate (expr. as N): 0.67 mgII Nitrite (elq)f. as N): 0.018 mgII Cyanide-totaal ( NEN 6655) <1.0 ugll Turbidity 0.049 FTE Colour intensity 20 mg Pt/I Dechlorination oftap water

### was notnecessary since no chlorine

### was present. "'Tap WII&erwas sampled fromthecopper-fnIe water supplysystemlhat supplied 1hewater used forthe presenttoxicity • B11Jdy. Among ethera, copperand badlJrfal activitywere measured ., ltIe6e samples.Theotheroomponentaware analysed In samples teken from Iha same waler supply butlIampted 11'1 the Animal House.

- Page 20 ­

## APPENDIX IV

96-HOUR ACUTE TOXICITY STUDY IN CARP WITH (FLOW-THROUGH); DETERMINATION OF THE CONCENTRATIONS

study comPletion date

October28. 2005



### Laboratory project Identification

- Page 1 of 15 ­1. CONTENTS 1. CONTENTS

...........................................................................................................................2 2. REPORTAPPROVAL

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

### 3 3. INTRODUCTION

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

### 4 3.1. Preface

...........................................................................................................................4 3.2. Aim 

### ofthe study

..............................................................................................................4 4. MATERIALS AND METHODS

...............................................................................................4 4.1. Reagents

........................................................................................................................4 4.2. Samples

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

### 4 

### 4.3. Analysis

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

### 5 4.3.1. Analytical method

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

### 5 4.3.2. calibration

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

### 5 4.3.3. Injections

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

### 5 4.3.4. Data acquisition and data processing

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

### 5 

### 4.3.5. Procedural recoverysamples

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

### 6 4.4. Interpre1atlon

..................................................................................................................6 4.4.1. calculations

.....'

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

### 6 4.4.2. Fonnulas

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

### 6 5. RESUlTS

...............................................................................................................................8 

### 5.1. Calibration curves...........................................................................................................

### 8 5.2. Samples

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

### 8 5.2.1. Procedural recovery samples

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

### 8 5.2.2. Test samples

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

### 8
TABLES Table 

### 1 Procedural recovery samples

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

### 9 Table 

### 2 Concentrations of. In test medium (range-finding test)

.............. 10 Table 

### 3 Concentrations of. in test medium (first limittest)

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

### 11 Table 4 Concentrations of. in test medium (second limit test)

................ 12 Table 

### 5 Results ofadditional attempts with changes in the

### flow-through system Intended to reach a higher exposure concentration**(~100** mgll) 

### in test medium

............................ 13
FIGURES Figure 1 GC chromatograms of a 0.0810 mgll calibration solution [lop; resJd. 

### 2205] and blank methanol [bottom; resjd. 

### 2217]
............................................... 14 Figure 2 GC chromatograms ofprocedural recovery samples at concentrations of

### 0.101 Figure 3 GC chromatograms of blank control samples taken during the first limittest att=O hours [top; res.ld. 1945], at t=4B hours [middle; res.ie!. 2248) and att=96 hours Figure 4 GC chromatograms oftarget 

### 221 mgn samplestaken during the first limit test 

### at t=­ 24 hours [top; res.ld. 1888]. 

### at t=O hours [second; res.id. 1913]. att=48 hours [third; 

### mgn [top; res.ld. 2235] and 101 mgJl [bottom; res.id. 2240]

......................................... 14 [bottom; res.ld. 2250]

.................................................................................................... 15
res.id. 2228] and at t=96 hours [bottom; res.ld. 2230]

................................................. 15

-Page2­

### 2. REPORT APPROVAL
PRINCIPAL SCIENTIST: (Analytical Chemistry)



### ....

Date:

# o(_:k~**l~** ,LGO;'

-Page3­3. INTRODUCTION 

### 3.1. Preface Study plan (analytical study) Start Completion : 30 May2005 : 05 October 2005 

### 3.2. Aim ofthestudy The purpose ofthe analytical study

### was to determine the actual concentrations in samples taken from·the test solutions used during theecotoxicitytest. 

### 4. MATERIALS AND METHODS 4.1. Reagents Methanol HPlC-grade. labscan, Dublin, Ireland Milli-Qwater Tap water purified by reversed osmosis and subsequently passed over activated carbon and ionexchange cartridges; Mlillpore Corp, Bedford, MA. USA ISO-medium see main report Tap water 

### see main report 

### 4.2. Samples All samples not analysed on the sampling daywere stored frozen. Stabilityofthe samples under these conditions was determined during "Developmentand validation of an analyttcal method 

### for the analysis of in Milli-Qwater". On the day

### of analysis, the frozen samples were defrosted at room temperature. The samples (10 ml) were analysed using headspace (see 4.3.1 analytical method).

- Page 4 •

### 4.3. Analysis 4.3.1. Analytical method Quantitative analysis was based on thetest substance peak in the GC chromatogram of**Ana~k8lcondfflbns** Column stationary phase Dimensions Brand Temperature program Initial temperature Initial time Rate 

### 1 Temperature 

### 1 Rate 

### 2 Final temperature Hold time Camergas Column 

### ftow Injection Temperature Splitless time Injection volume Injection speed Headspace conditions Sample Incubation temperature Incubation time Syringe temperature Detectfon 4.3.2. Calibration CP Porabond Q 

### 25 m x 

### 0.25 mm, 

### dt

# = 3 IJm Varian, Mulgrave, Vletorla, Australia


### 40"C 

### 1 min 20 "C/mln 

### 190 

## "c 50 "C/mln 

### 250°C 

### 3.3 min Helium 1 mllmin Splitress 

### 250"C 2 minutes 

### 500 loll (Headspace) 50 IJI/sec


### 10 ml ofan aqueous sample in a 

### 20 mlvial 

### 5O"C 30 minutes 

### 110°C FlO (250 "C; Hydrogen: 40 mVmin; Air. 450 mflmin)
During the rangefinding test, calibration solutions in ISO-medium were made upfrom two standard solutions of In methanol. During the final

## test. callbratlon solutions in tapwater were made up

### from two standard solutions of in methanol. 4.3.3. Injections Calibration solutions were injected in duplicate. Procedural recovery samples and testsamples were analysed bysingle InJectlon. 4.3.4. Data acqulsltlon and data processing Data acquisition and data processing was perfOlmed using Empower software (Waters. Milford, MA, USA)version 5.00.
-Page 5­

### 4.3.5. Procedural recovery sample. Standard solutions of In methanol and dilutions ofthese solutions with methanol were used as spiking solutions.**Durin~** the range-finding test, ISO-medium 

### (10 ml) was spiked at concentration levels of 

### 0.101 mgll and 

### 101' mgll using 

### 10.1 mgll and 

### 10085 mg/l spiking solutions in methanol. During the first limit test on 

### 28-06-05 and 

### 29-06-05, tap water 

### (10 ml)was spiked at concentration levels of

### 0.101' mgll and 

### 101' mglJ using 

### 10.1 mglJ and 

### 10150 mgll spiking solutions In methanol.**Durin~** the first limittest on 

### 04-07-05, tap water 

### (10 ml)was spiked atconcentration levels of 

### 0.101 mg/l and 

### 101' mg/l using 

### 10.1 mgll and 

### 10119 mgll spiking solutions In methanol. During the second limittest on 

### 13-09-05, tap water 

### (10 ml) was spiked at concentration levels of 

### 10.1' mg/l and 

### 100' mgll using 

### 1013 mgll and 

### 10011 mgll spiking solutions in methanol. During the second limit test on 

### 19-09-05, tap water 

### (10 ml) was spiked at concentration levels of 

### 10.0' mgll and 

### 100' mgll using 

### 1013 mgll and 

### 10011 mgll spiking solutions In methanol. During the additional test, tap water 

### (10 ml) was spiked 

### at concentration levels 

### of 

### 10.0' mgll and 

### 100' mgll using 

### 1001 mgll and 

### 10009 mg/l spiking solutions in methanol. During the range finding test and first IimH test, at 

### 0.101 mgll one and at 

### 101 mg/l five samples • were prepared. During the second limit test and the additional test, three samples were prepared ateach concentration level. All recovery samples were treated as described in paragraph 4.2 'Samples' and analysed. The recovery was calculated for each sample


### 4.4. Interpretation 

### 4.4.1. Calculations All calculations 

### were performed using non-rounded values. Rounded values are reported In the tables. Therefore, some differences might be observed when calculating the parameters as mentioned In the tables. : 

### 4.4.2. Fonnulas General Mean:
where Xl = measured value n 

# = number 

### of measurements

n 

### L{Xi-X)2 Standard deviation Sn - 1= U....:::1-1'-:-----:_ en-I)
Coefficient ofvariation

1 ConcenlraUons were not corrected forspiking volume

- Page 6­Calibration Response: R 

### = Peak area test substance [unlts1 Calibralion 

### curve: The response 

### was correlated wIIh the test substance concentration, using linear regression analysis Qeast squares method; weighting factor (1/concentration2)}.

R = response calibration solution [units] C 

# = concentration of

### testsubstance in calibration soiutlon [mg/l] a 

### = slope [unlts*lImg] 

### b 

### = intercept [units] Samples Concentration 

### ofASAHIKLIN AE-3000 analysed In the samples: C '" 

### (R -b) *d [mgll} 

### a R 

### = response sample [units] d 

### = dDution factor a 

# = slope [unlts"'lfmg] b '" intercept [unHs) Recovery ofprocedural recovery samples: Concentration analysed

... 100 

### rk} Concentration prepared Concentration analysed '" 

### 1 00 [%] Relative to nominal concentration: Concentration nominal
Concentration analysed at t • x hours Relative to Initial concentration: 

### '" 100 

### [%J Concentration analysed 

### at t == 0 hours



### -page7­

### 5. RESULTS 5.1. Calibration curves 

### On each day ofanalysis, a calibration curve was constructed using at least six concentration levels. 

### Ateach concentration level, two 

### responses 

### were used. The coefficient

### ofcorrelation was in excess 

### of0.99 for each calitXation curve. Figure 1shows chromatograms of

### a calibration solution and blank methanol. 

## 5.2. Samples 5.2.1. Procedul'IIl 

### recovery samples Figure 2 shows chromatograms of procedural recovery samples. The results forthe 

### procedural recovery samples are summarized in Table 1. Mean recoveries of

### procedural recovery samples are required 

### to be 

### in the range 70-110%. Because the recoveries 

### ofthe procedural recovery samples were met, the results 

### ofthe

### test samples

### were considered accurate. 5.2.2. Testsamples Figures 3 and 4 showchromatograms of blankcontrol samples and target221 mgll samples taken during the first limit test. The results forthe test samples are summarized in Tables 2 - 4. During the range-finding test, the test-substance concentrations if1 

### test samples 

### ofnominal 10 mgll taken at t=0 hours were below

### the limit

### ofquantlftcaUon (LOQ) of

### the analytical method, but withIn the calibrated concentration range olthecalibration solutions. Because the

### coeffICient ofcorrelation ofthe calibration curve

### was in excess of 0.99 the 

### results ofthe 

### test samples 

### were considered accurate. The concentrations ofthe 

### test samples 

### att=96 hourswere significantly below the concentration ofthe Ipwest calibration solution, therefore < 

### LOQ 

### was given. During the first limit test, 

### a small peakatthe retention time ofthe

### testsubstance was observed In the chromatograms ofthe blank test samples (nominal 0 mgll) 

### att=48 and F96hours. Because a similar peakwas found in blanks of

### methanol 

### itwas concluded thatthe compound 

### did not derive from 

### test medium. Forthe blank test samples, the response ofthe interference was smallerthan the response ofthe lowest calibraUon solution, therefore < 

### LOQ was given. Forthe test 

### samples oftarget 

### 221 mgll, the maximum contribution of

### Ifle compound to the peak area ofthe lowest 

### test sample was 0.2%, and was therefore notconsidered to be 

### a significant Interference. During the second limittest, 8 

### small peak atthe retention time ofthe test 

### substance 

### was observed in the chromatograms ofthe blanktest samples (nominal 0 mgll) at t=48 

### and t=96 hours. This peak

### was probably caused by a carry-over effect 

### ofthe analytical system. The maximum contribution ofthis peak

### to the samples 

### oftarget 221 mgll was 

### 0.08%, and was therefore notconsidered to be a significant interference. In Table 5, the results 

### ofadditional attempts with changes in the fIow-through system intended 

### to reach 

### a higherexposure concentration**(~100** 

### mgll) in test medium are shown.

-PageS.TABLES 

### Table 1 Procedural recovery .ampl

.. 

### Date of 

### preparation [dd-mm·yy] Date 

### of analysis [dd-mIn*YY} Concentration nominal

[mgJl) Concentration analysed

[mgIIJ Recovery 

### [%] Mean recovery (Coefficient

### of

variatIon) 

### r%1 

### 30·0S"()5 ' 30..()5..()5 0,101 0.109 108 3Q..()5-05 1 

### 30..()5..()5 101 101 101 101 101 98.6 

### 79.1 10S 100 74.2 98 

### 78 104 

### 99 74 91 (1S) 28..()6...(}S' 

### 28..()6...(}5 

### , 0.101 0.109 108 28..Q6...(}5' 

### 28...(}6...(}5 101 101 101 101 101 99.7 99.4 99.0 102 100 98 

### 98 98 101 99 99 (1.3) 29...(}6...(}S ' 29...(}6...(}5 0.101 0.0813 2 80 29...Q6..0S' 

### 29...(}6...(}5 101 101 101 101 101 97.6 96.7 96.3 94.S 94.8 

### 96 95 95
. 93 93 

### 95 (1.3) 04...Q7-05 ' 04...Q7.05 ' 04"'(}7..Q5 04-07-05 0.101 101 101 101 101 101 . 0.0989 2 

### 86.0 92.S 87.7 91.4 95.0 98 85 

### 92 87 90 94 

### 90 (4.1) 

# .

13-O9...(}5 13-09...(}S



### , 10.1 10.1 10.1 10.6 10.4 10.8 104 103 107 105 (2.0) 13-O9"()5 13-09-05 100 100 100 110 112 111 110 112 111 111 (0.79)
19..Q9"()5 19..()9...(}5 10.0 10.0 10.0 10.4 8.06 

### 10.1 104 81 

### 101 95 (13.3)



### - Page 9·2 

### Table 1 Continued 

### 19"()9-O5 19-09-05 100 95.0 95 82 100 73.2 73 (13.7) 100 79.0 79 03-1Q..()5 03-1Q..05 10.0 10.0 100 100 10.0 10.1 101 (0.46) 10.0 10.1 101 03-10-05 03·10-05 

## I 100 92.2 

### 92 91 100 89.9 90 (1.3) 100 91.3 91 04-10-05 04-1Q..05 10.0 10.1 101 100 10.0 10.0 100 (0.56) 10.0 10.0 100 04-1Q..()5 04-1Q..05 100 97.5 97 96 100 97.3 97 (2.5) 

# . 100 93.2 93 Combined with Concentration

_ below the limit of quantification. butwithin theconcentration range ofthe calibration solutions.


### Table 2 Concentrations of In test medium (range-finding test)



### Time of 

### sampling

[hours) Date of sampling [dd-mm-yy] Date of analysis [dd-mm-yy] t 

### Concentration Nominal

(mgll] 

### Analysed [mgll] 

### Relative to nominal

[%] 

### Relative to mean Initial [%1 

### 0 23-05-05 3Q..05-05 10 10 100 2 

### 100 2 

### 0.0822 3 

### 0.0871 3



### 3.71 6.58 1 1 4 

### 7 24 24"()S-OS 30-05-05 10 10 100 100 0.234 0.245 4.77 5.34 2 2 5 5 276 290 93 104 96 

### 27-05-05 30-05-05 10 10 100 100 <LOO <LOa



### 1.16 0.969 

### 1 1 22 19 Samples were frw:en unlll analysis. Combined with Concentndion was below Ihe limit ofquantification, butwithin the concentration range ofthe calbration solullons. LOO LimitofquanlOcatlon was 0.100 mgIl "Developmentand validation ofan analytical method b"the

## ;marys,- of ' In MIlII-Q water").



### - Page 10­

### Table 3 Concentrations of In test medium 

### (first limittest) Concentration**~ate** 

### of 

### Date of 

### Time of Relative to sampling sampling analysis Target Nominal Analysed nominal [dd-mm-yy] [hours] [dd-mm-yy1 [mgll] [mgJI] 

### (mgllJ [%] 

### 36 •**~24** 

### 100 28-06-05 28-06-05 221 80.2 221 100 87.9 40**29~06-05** 

### 29-06-05 

### 0 0 n.d. n.a. 

### 0 

### 0 0 n.d. n.a. 

### 221 100 81.0 37 , 

### 100 221 87.7 40 0 48 01-07·05 04-07-05 ' <LOQ 2 

### 0 n.a. 

### 0 <LOQ 2 

### 0 n.B. 

### 221 100 

### 84.2 29 i 

### 221 100 59.9 27 03-07-05 0 96 04-07-05 1 <LOQ 2 

### 0 n.a. 

### 0 <LOQ 2 

### 0 n.s. 221 

### 100 SO.5 

### 27 i 

### 221 100 i 84.6 

### 38 i i Sampleswere fnIzen untiIlnllysis. lOO 2 AIImII peakwas obseMICI at\tie retention limeofteet$Ub8t8nee, but lis reaponse was not considered

### to derive fromtest sample $Inee ItWIIS also observed In blank methanol. Limit ofquantlllciltlon WIll 0.100 mgII (See :·Development and vandalion ofan analytieal method fDr\tie anary.of In Mnll-Qwater1. n.d.**Not~** n.a. Not applicable



### • Page 11

­

### Table4 Concentrations 

### of In 

### test medium (second limittell) Time of sampling

[hours} Date of sampling [dd-mm-yy] Date 

### of analysis {dd-mm-yy] Concentration Nominal

[mgll] Target [mgll] Analysed {mgll} Relative 

### to nominal

[%] 

### -1



### 48

96 

### 13-09-05

15-09-05

17-09-05 13-09-05

19-09-05 1



### 19-09-05 1 

### 0 0 221 221

0 0 221 221



### 0 0 221 221 

### 0 0 

### 100 100 0 0 

### 100 100 0 0 

### 100 100 n.d. n.d. 

### 85.4 85.6

2 

### z 60.6 72.5

41 2 

### 82.9 69.1 

## n.a. n.a. 39 

### 39 n.s. n.a. 

### 27 

### 33 

### n.a. ".a. 38 

# . 31 Samples

### werefrozen until .nalysls. A sman peakwas obs8M!Id inU- nominal 0 my/!samplet. This Milprobablycaused bya C8I"1)'-OW!efI'ed of the analytical aystem•.The maximum conIIbJtIon ofthis peakto the sampleswasOJ16%. n.d. Not dtIteded n.a. Not applcable

- Page 12­Results ofadditional attemptswith changes inthellow-through system 

### intendedto 

### reach a higherexposure concentration**~100** rngIl) intest 

### medium Table 5


### i Date of Date of sampling analysis [dd-mm-yy] [dcknm-yy) 

### 30-09-05 

### moming 30-09-05

03-10-05' 30-09-05 afternoon



### 04-10-05 

### morning 04-10-05

04-10-05 04-10-05 afternoon Nominal 

### [mgll]

, 

### 0

0 0 176 176 176 176 e 

### 176 

### e 176 7 

### 176 8 

### 176

### s 176 6 

### 176 7 

### 176 8



### 0 0 0 176 176 176 265 265 

### 265 

### , Target 

### [rng/l]

0 0 0 100 100 100 100 100 100 100 100 100 100 100 0 0 0 100 100 100 100 100 100 Concentration Analysed



### [mgll]

2 2 3 2 2 

### 64.2 4 

### 20.1 50.3 56.7 59.1 45.1 56.1 36.6 

### 56.5 n.d. n.d. n.d. 

### 46.1 47.1 47.5 73.6 72.9 73.8 Relative 

### to

## ~t n.l. 

### n.s. n.a. n.a. n.a. 

### 36 11 

### 29 

### 32 34 26 32 21 

### 32 

### n.l. n.s. n.a. 26 27 27 28 28 28 24 64.2 100 265 

### 05-10-05 05-10-05 26 100 68.5 265 

### 26 100 69.6 265 Samplea W8rafrozen until analy8is. Due to problems with the analytical system itwas not P05llbie 10determine an analysed concentration. Due 1.0 problems witt! !lie analytbllsystem this sampleWItS notanalysed. IndlcatM!ll8SUl. Bottom sample (from) Bottom sample (back) Top sample (front) Top 8IW11ple (back) n.d. Not detected 

## n." Not applicable



### -Page 13­FIGURES



# .... 

# ... 

# . 

# .. .... 

# ..

# .. 

# ... Figure 1 GC chromatograms ofa 0.0810 mgll calibration solution 

### [top; resold. 2205] and blank methanol 

### [bottom; res.ld.2217]•



# .... 

# ... 

# . 

# .. I

.. 

# ... ..

# .. ..

# .. ..... Figure2 GC chromatograms of procedural recovery samples

### atconcentrations 

### of 0.101 mgn [top; res.ld. 2235] and 101 mg/l [bottom: res.ld. 2240],



### - Page 14­

# ! 

# I ·1



# I ...a 

# ... Figure 3 GC chromatograms of blank control samplestaken during the first limittest att-O hours [top:

.....Id. 

### 1943]. att-48 hours [mlddla:

.....Id. 2248] and at 

### 1-98 hours [bottom; ra• .ld. 2250]

.



# .**:~ .~1--**  

# k**-------------**

# .~~------I-'-2r- 

# L**.~ut------~-t-':!r--**



# l

....

# ~~~ , , 0 

### , i

# .~~ ilO 

# ... 

# ... .i. ,II

# ~

# l

.. .... 

# ... 11,. 

### "

... i FIgure 4 GC chromatograms oftarget221 mgn samplestakan during thefirst limit testat (=-24 hours [top; res.id.1888], at

### t=o hours [second; res.ld. 

### 19131. at 

### t-48 hours [third; ras.ld. 2228] and att=96 hours 

### [bottom; res.id. 

### 2230].

- Page 15­

## P-04..635

. REPORT

StudyTitle 

## ACUTE TOXICITY STUDY IN 

## DAPHNIA MAGNAWITH (FLOW-THROUGH)

Study completion elate



### 16 September 2005
Test Faclltlv

Laboratory Project Identification

• Page 1 of 32 ­1. 

### CONTENTS 1. CONTENTS

...........................................................................................................................2 

### 2. STATEMENT OF GLP COMPLIANCE

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

### 3 3. QUALITY ASSURANCE STATEMENT

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

### 4 

### 4. SUMMARY

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

### 5 5. INTRODUC·nON

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

### 6 5.1. Preface

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

### 6 5.2. Aim of the study

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

# e 5.3. Guidelines

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

### 6 

### 5.4. Storage and retention of records and materials

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

### 6 5.5. Oeftnltlons

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

### 7 

### 6. MATERIALS AND METHODS

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

### 7 6.1.1. Test substance Information

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

### 7 6.1.2. Reference 

### tast
......................................................................................................

### 7 6.1.3. Preliminarydata

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

### 7 

# :I**~=r"s~.:::::::::::::::::,::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::** 6.4. Range-finding test (static)

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

### 9 6.5. First flow-through test
.....................................................................................................

### 9 

### 6.6. Final flow-through test
....................................................................................................

### 9 6.6.1. Concentrations

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

### 9 6.6.2. Dosing system
.................................................................................................... 10 

### 6.S.3. Test procedure and conditions

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

### 11 

### 6.6.4. Sampling for analysis oftest concentrations

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

### 11 6.6.5. Measurements and recordings

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

### 11 6.7. Interpretation

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

### 11 6.7.1. Data handling

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

### 11 6.7.2. Acceptability of thetest
...................................................................................... 

### 12 6.S. List of protocol deviations

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

### 12 7. RESULTS

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

### 12 7.1. Range-finding test
........................................................................................................ 12 

### 7.2. First limit flow-through test
........................................................................................... 

### 13 7.3. Final flow-through test
.................................................................................................. 

### 13 7.3.1. Measured concenlrations

................................................................................... 13 7.3.2. Immobility

........................................................................................................... 14 

### 7.3.3. Determination of effect concentrations

.............................................................. 15 

### 7.3.4. Experimental conditions

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

### 15 S. CONCLUSION

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

### 15 TABLES Table 

### 1 Dosing program.................................................................................................. 

### 10 Table 

### 2 Incidence of immobility in the range-tlnding tesL.

............................................. 12 Table 

### 3 Incldence of immobility 

### In the limit 

### test
............................................................... 

### 13 Tabla 4 Mean measured concentrations versus nominal concentration

........................ 14 TableS Acute immobilisation of daphniaafter

### 24 and 

### 48 hours In the final test
............ 

### 14 TableS Effect parameters
............................................................................................... 15 Table 

### 7 pH and oxygen concentrations during the final test.
.......................................... 15 FIGURES Figure 1 Row-through systElfn

.......................................................................................... 10 APPENDICES APPENDIX I REFERENCE TEST

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

### 16 APPENDIX II ANALYSIS OF TAP WATER (Not performed under GLP) APPENDIX III ANALYTICAL REPORT ................................ 

### 17

-Page2·2. STATEMENT OF GLPCOMPUANCE

The studydescribed in this report has been correctly reported and was conducted in compliance 

### with: The Organization for Economic Cooperation and Development (DECO) Good Labormory Practice Guidelines (1997). Which essentially conform to: The United States Food and Drug AdmInistration Good Laboratory Practice Regulations. The United States Environmental Protection Agency Good Laboratory Practice Regulations. Analyses oftap water was excludedfrom this GlP statement. The sponsor is responsible for Good Laboratory Practice (GlP) compliance for all test substance Information 

### unless determined by

Study Director Head of In Vitro & Environmental Toxicology



## Tectftcal DIrector

Date:

..

# 19.I.O.S

...**~g5** 

### ..........-...........

- Page 33. QUALITY ASSURANCE STATEMENT

This report was inspected 

### bythe QualityAssurance Unit to confirm that the methods and results accurately andcompletely reflectthe raw data. The dates of QualityAssurance Inspections are given below. During the on-site process inspe<:1ions procedures applicable to this type of studywere Inspected.

Type

# 0' Inspections PhaselProcess ' 

### Start 

### Inspection date End Inspection date Reporting data Study Protocol ()3.-Sep-04**O~Sep4i** 03-Sep-04 Amendment 1of protocol 03-May"()5 03-May-05 

### 03-May-05 Amendment 2 of protocol 27-May-OS 27-May-05 27-May-05 Amendment 3 of protocol 22-Jun-05 22-Jun-05 22-Jun-05 Amendment 4 of protocol 30-Jun-05 3O-Jun-05 30-Jun-05 Amendment 5 of protocol 19-Jul-05 19-Jul-05 19-Jul-05 Amendment 6 of protocol 22-Jul-05 22-Jul-05 22-Jul-05 Report 01-Sep-05 01-8ep-OS 01-8ep-05 Process Environmental toxicology 1B-Apr-05 22-Apr-oS 22-Apr-OS Test substance handling Exposure Observations/Measurements Analytical and physical chemistry 2O-Jun-05 29-Jun-OS 05-Jul-05 Test substance handling ObservationslMeasurements

Head of Quality Assurance

-Page4­

### 4. SUMMARY Acute Toxicity Study in 

### Daphnia magna with The study procedures described in this report were 

### based on the ISO International Standard 

### 6341. 1996. In addition, the procedures were designed to meet the test methods Bnd validity criteria of the EEC directive 

### 92169, Part C.2. 1992 and

## the OECD guideline No. 202 Part I, 1984. The batch of lested was a clear and coIoutless liquid with a purity of 99.985%. was completely soluble in test medium at the concentrations tested and known 

### to be extremely volatile. Consequently, measures were taken during this project

### to minimise 

### loss of 

### test substance by evaporation, which included butwere not limited to short preparation times and the use of airtight closed vessels. The project started with a statiC range-flndlng test exposing ten daphnia per concentration (In duplicate, 5 pervessel) toa blank-control and nominal concentrations of 0.1. 1.0, 10 and 100 mgll. No significant Immobilisation of daphnlds was observed at any of the concentrations tested. Analytical results of samples taken from 100 mg/l showed that the mean measured concentrations were stable at approximately 5 mgll during the 48-hour test period. The project was continued with a limit tes1 in an alr.tlght closed flow-through system with continuous renewal oftest solutions. Twenty daphnia per concentration (four meshes, 5 daphnia per mesh) were exposed for 48 hours to a blank-control and a nominal concentration 

### of 221 mg/!. Which was expected to correspond to an actual exposure concentration closeto 100 mg/l. Precipitate in the form of droplets and floating daphnia Influenced the results 

### of this

### test by mechanical disturbances. Therefore, the results were considered not representing the intrinsic toxicity of the lestsubstance. Itwas consequently decided to continue with afinal flow-through studytesting a range of concentrations. The final test was performed in an air-tight closed flow-through system with continuous renewal of testsolutions. Exact volumes of pure test substance were dosed every 5 minutes and mixed with tap wateral a flow rate of 12 LIh. Twenty daphnia per concentration (four meshes, 5 daphnia per mesh) were exposed for48 hours to a blank-control and nominal concentrations of 22, 40, 71, 123 and 221 mgll. It was anticIpated that this nominal concentration range would correspond to a range of actual concentrations between 10 and 100 mgll. Samples for analytical confirmation of actual exposure concentrations were taken one day before onset, at the start and after

## 48 hours of exposure. Analyses of tt1e samples taken trom the lowes1 and highest test group one day before thestart of the test showed that 

### the system functioned property and thai the actual concentrations were close tothe anticipated targets. The mean measured exposure concentrations were relatively stable at the 

### two highesttarget concentrations and In agreement with what was expected. More variation was seen In the concentrations measured in the three lowesttest groups. However, only the highest exposure concentration was essential for determination ofthe toxicfty parameters and corresponded to 94 mg/l. The siudy metthe acceptability criteria prescribed by the protocol and was considered valid. The 48h-ECso exceeded a mean !11easured concentration of 94 mgl1 underthe conditions of the • present flow-through studywith 

### Daphnia magna exposed to

-Page5­ 

# ; !

# I**~-.~--".------.--** 

### ...5. INTRODUCTION 

### 5.1. Preface Sponsor

Study Monitor Test Facility

Study Director Technical Coordinator Principal Scientist Study Plan Start Complstion : 23May2005 : 22 July 2005 5.2. Aim ofthestudy The purpose of the studywas to evaluate the test substance for its abilitytogenerate acute toxic effects on the mobllity of 

### Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC!IO at 

### 24 and 

### 48 hours of exposure. 

### 5.3. GuldellMs The 

### study procedures described in this report were based on the ISO Intemational Standard 6341: "Water quality· Determination of the Inhibition ofthe mobility of Daphnia 

### magna StrausAcute toxicitytest. Third edition, 1996-04-01. In addition, the procedures were designedto meet the test methods.and validity of the following guidelines:

. . • European Economic Community (EEC), EEC directive 

### 92169, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, 0.2. "AcuteToxicityfor 

### Daphnia", • Organization for Economic Co·operation and Development (OEOD), OECO guidelines for Testing of Chemicals, guideline No. 202 Part I: 

### "Daphnia sp., Acute ImmobilisationTest", Adopted April 4, 1984. 5.4. Storage and retenUon of records and materials Records and materials pertaining to the study including protocol, raw

### data, specimens (except specimens requiring refrigeration orfreezing) and the final report are retained in the archives for a period of at least 10 years afterfinalization of the report. Afterthis period, the sponsorwill 

### becontacted to determine whether raw data and specimens should be returned to them. retained or destroyed on their behalf. Those specimens requiring refrigeration orfreezing will 

### be retained by for 

### as long as the quality ofthe specimens permits evaluation but no longerthan three mon1hs afterfinalization of the report.

• PageS­will retain a test substance sample until the expiry date, but no longerthan 10 years after finalization ofthe 

### report. After this period the sample will 

### be destroyed. 

### 5.5. Definitions Immobileare those animals not able to swim wllt'lln 

### 15 seconds after gentle agItation of the lest vessel.

.The ECso Is the concentration oftest substance estimated to immobllise 50% 

### ofthe daphnia after a defined period of exposure. No Observed Effect Concentration (NOEC) Is the highestconcentration tested 

### at which no effect (I.e. immobllisatlon) Is recorded. A Flow-through 

### test is 

### a test In which the water containing the test substance is renewed continuously.
6. MATERIALS AND METHODS 

### 6.1.1. Testsubstance InformaJion Identification Structure
Molecularformula Molecularweight CAS Number Description Clear colourless liquid Batch Purity 

### 99.985% Test substance storage In refrigerator in the daM< Stability understorage conditions 

### Stable Expiry date 28 February 2006 Specific Gravity 

### 1.47 Stability in water Not indicated 6.1.2. Reference tasl This report includes the results of the most recent reference test with potassium dichromate (Appendix I). 

### 6.1.3. PrelimInarydata The water solubility of at 

### 20.1 ;I; 

### OSC is 

### 0.805 gil using the flask method

. Further essential information obtained from physico--ohemlc:al studies performed at were 

### the vapour pressure being 

### 31 kPa at 

### 25°C, freezing poInt 

### <-81°e, boiling 

### polnt'56"C and a log 

## Koc of 2.54. The 

### log Pow of 

## was 2.18 

### accordlrlg to informatIon supplied by 

### the sponsor. From these data it can be concluded that thetest substance will evaporate rapidly at room temperature as pure substance but also from aqueous 3 1 solutions (calculated Henry coefficient is 

### 7.24.103 

### Pa m mor ). concentrations showed to be stable during freezing and thawing of samples in a deep-freeze

-Page7­

### 6.2. Test ayslem Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), 

### atleastthird generation, obtained by aeyclfcal parthenogenesis under specified 

### breeding conditions. 

### Source In-house laboratory culture with 

### a known 

### history. Reason for selection ThIs system has been selected as an internationally accepted Invertebrate species. to 5111 Validity of batch Daphnids originated from a healthy stock.**~** brood. showing no signs of stress such 88 mortarlty >20%, presence of males, ephlppia ordiscoloured animals and there was no delay in the production ofthe first brood. 

### Characteristics Forthe test selection ofyoung daphniawith an age of < 24 hours, from parental daphnids of more than two 

### weeks old. 

### 6.3. Breeding Start 

### of each batch With newborn daphnids, I.e. less than 3 

### days old, by placing about 250 of them Into 5 litres of medium In an alliJlass culture vessel. Maximum age of 

### the cultures 4 weeks Renewal of the cuHu'res After 7 days of cultivation half of

### the medium twice a week. Temperature of medium Feeding Daily, asuspension offresh water**alga~.** Medium M7, as prescribed by

### Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage In Daphnia magna Straus. Protoplasma 154,25-33). Qomposltfon of medium 

### MZ: ISO medlm1: Ihe

### roIowtnvchemICal. (analytical grade) are dI1l8oIved In 18pwater purified by R9V9rs8 OSl1'lOllis (Mllt-RO, MIIlipore Corp., Bedford. Mass., USA): Macro salts:**CaCI~** 293.8 mg/! MgSO••7HtO 123.3 mgll NaHC03 84.B mgll KCI 6.8 mg/! Medium M7: tmce C!lements. macro nuttienlll and vitamins are added to freshly prepared ISO medltm 10 reach the following concentJatlons: Trace elemenls: B 0.125 

### mlf! Fe 0.05 TIl!o)1 Mn 0.025 mgII LI. Rbend Sr 0.0125 

### ffi9II Me 0.0063 TIl!o)1 Br 0.0025 mgII CU 0.0016 mgll -PageS­Zn 0.0063 

### mlVl Coend I 0.0025 moll Be 0.0010 mgJ V 0.0003 

### mlil NaaEDTA.2H.o 2.5 

### mgll Macro nutrients: NaaSiOa. 9HtO 10.0 

### mgtl NaNOa 

### Ozl mgtl KH.tPO. 0.14 

### m(VI 

### KaHPO. 0.18 

### mlVl VItamins: Thiamine 75.0 pg/I Bu 1.0 

### WI Biotin 0.75 pg/I Thehardr'IesB: 250mgI1 expressed 

### as Caeo,and the pH: 8.0:1:0.2after aemtlon. 

### 6.4. Rang
..flndlng test (static) A static range-finding test was performed to provide information about the range of concentrations to be used In the final test. Test procedure and conditions were similar

### tothose applied In the final test with the followlng exceptions: a static test system 

### was used In which ten Daphnia per concentration (two 

### vessels, five pervessel) 

### were exposed to a blank-control and nominal concentrations of 

### 0.1 to 

### 100 mgJI increasing by a factor of 10. Dissolved oxygen concentrations, temperature and pH were only measured in the control and highest test concentration. Duplicate samples for possible analyses were taken from all test solutions at the 

### start (in combination with and after

### 48 hours of exposure. Preparation forthe range-finding test started with test solutions prepared at 

### 10 and 

### 100 mgll 

### applying' or' 

### 3 minutes of magnetic stirring, respectively. Care 

### was taken that during preparation 

### as little as 

### possible volatinsation occurred (weighing and preparation 

### in closedvessels which already contained 

### a sman volume of test medium, short preparation period). The lower test concentrations were prepared bydilution of the test solution of 10 mgll. The tlnal test solutions were aU clear and colourless. The test solutions originated from those used for 

### the simultaneously performed acutefish toxicity 

### test 6.5. First

### flow-through 

### test The project was continued with a limit test underflow-through conditions. The nominal test concentration was 

### 221 mg/l, which was expected to correspond to an actual exposure concentration close to 100 mgll. A blank-control was Included. Test procedure and conditions were similarto 1hos9 applied in the final flow-through test. 

### 6.8. Final flow-through test 

### 6.6.1. Concentrations Nominal 

### 22, 40. 71, 123 and 

### 221 mgJ1. expected to correspond to actual concentrations close to 

### 10,18,32, 56 and 

### 100 mgJI. Controls Test medium without test substance 

### or other addi1iv9s (blank-control)•

• Page 9­6.6.2. Dosing system SuffICient amounts of pure test substance were collected daily in airtight sea/ed vials and used as 'stock' solutions for dosing. The pure test substance was 

### dosed vie a computer-controlled system in combination with 5 dispensers (GUson). Via this system the 

### dosed volumes entered a mixing flask separatelyfrom the tap watersupply. The

### tap waterwas supplied via a flow meter

### wfth a flow rate ofca. 12 1Ih. In the mixing flask the 

### dosed volumes and \he tap water were mixed under continuous stirring. The whole system was checked dally. The dosing program is described In Table 1 and a diagram of the system is presented In Figure 1. The hourly dosed volumes in combination 

### wfth atap-water flow of 12 liters per hour resulted in a nominal concentration range between 22 and 221 mgll. However. anticipating the rapid loss of test substance, it was expected thatthis dosing regime would give an actual concentration range between 10 and 100 mgII (see the last column in table 1). Table 1 Dosing program

STOCK SOLUTION DOSING VOLUMES (0.6 mt 

### syrlngel NOMINAL CONCENTRATION EXPECTED CONCEHTRAnON PERCVCLE PER HOUR pl/5

### min ml I 

### hour (mg/I)**(~)** 15 0:18 22 10 

### 27 0.32 40 18 Pure 48 0.58 71 32 84 1.0 123 58 150 

### '.8 221 100 Diluter 

# 0 1 +­ Computer 

# 1 ap-water



# ..
Test ¥essel

Figure 1 Flow-through system

-Page10­6.6.S. Test procedure and conditions Test duration 48 hours Test type Flow-through, continuous renewal of

### test media. Test vessels ca. 

### 1.5 litres, stainless steel containing four stainless steel meshes and airtight closed 

### with a Perspex lid. Medium Tap water (see Appendix II for data ofanalysis) Numberof daphnia 

### 20 perconcentration Loading 5 per mesh Ught 

### 16 hours photoperiod daily Feeding No feeding Aeration No aeration ofthe test solutions. Introduction of daphnia Two days afterthe start 

### of the dosing 

### to permit stabilisation of the system 

### 6.6.4. Samplingforanalys's 

### 01

### testconcentrations During the final test duplicate samples for analysis were taken according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix III). Sampling: Frequency At ca 

### 27 hours following the start of

### dosing, samples were taken from nominally 22 and 221 mgIJ to check the actual exposure concentrations. At the 

### start and 

### the end of the exposure 

### period samples were taken from all concentrations andthe cootrol. Volume 

### 10 mlfrom the approximate centre ofthe 

### testvessel. Storage Samples taken at the end ofthe test 

### were stored in a deep-freeze until analysis. 6.6.5. Measurements and**~rdlngs** Immobility pnofudlng mortality) At24 hours and at 48 hours. pH and dissolved oxygen At the beginnIng, after 24 hours and atthe end of the test. for all concentrations and the control. Temperature of medium Continuously in the vessel containing the blank-control, beginning at the start ofthetest. 

### 6.7. Interpretation 6.7.1. Data handling Calculation of ECso: No 

### ECeo could be calculated (EC60 > maximum concentration tested)

.

• Page 11 •

### Defining exposure cOllCentrations: The results were based on the mean of the concentrations measured in samples taken at the 

### various time Intervals. 6.7.2. AcceptabllHy 

### ofthe

### test 1. 

### In the control, no

### daphnia became immobilised ortrapped 

### atthe surface of (he 

### water. 2. The oxygen concentration was ;;e 

### 5 mg/l at the end of

### the test. Other test conditions (pH and temperature) were maintained within the 

# 1mits 

### prescribed bythe guidelines. 3. 

### The 24h-ECso (based

### on the initial concentration) of 

### potassium dichromate was within the 

### range 0.6 mg/l 

### to 1.7 mg/l. 

### 6.8. L1at of

### protocoldeviations There were no deviations from the protocol.


### 7. RESULTS 

### 7.1. Range-finding

### test Table 2 shows the 

### responses recorded during the static range-finding test No significant immobHisation of

### daphnjds was observed at any of the concentrations tested. Analytical results of samples taken from 100 mgll showed that the 

### mean measured COllCentrations W&l'e stable 

### atapproximately 5 mg/l during the 48-hour

### test period 

### (see Table 2 of the appended Analytical report). Hence, 

### with the current preparation procedure actual exposure collCentrations close to 

### nominal could not 

### be reached. To enable tasting higher actual collCentrations and 

### to increase the security of maintaining these stable It 

### was decided to continue testing applying aflow-through system. All further test conditions were maintained within the limits prescribed by the protocol. Table 2 IncidenceofImmobility In the range-finding 

### test

Concentration 

## v..... Number Response

### at24 h Response

### at 48 h number Daphnia Total Total expoMd number % number % Nominal (mgll)

Blank-conlrol A 5 1 

### 5 1 5 B 5 

# om 0 0.1 A 5 0(3) 0 0(2) 5 B 5 0(4) 

# I 1(5) 1.0 A 5 0(5) 0 0(3) 0 I B 5 0 0 

### I 10 A 5 0 0 0 0 

# I B 5 0 0 I 100 A 5 0(3) 0 0(5) 0 

### , B 5 

## o(1) 

# I 0 Between bracI<8Ia: numberd daphnlds observed trapped at 1I1e surface. These organisms

__ relmmEu'sed InIO the 1'86p&Ctive solutIonS before recording ofmobility,

-Page 12­·7.2. First lIm1t flow-through test Table 3 shows the responses recorded during the limit flow-through test. Atotal of 80% of the daphnids exposed to 

### the limit concentration were immobllised after

### 48 hours of exposure. Analyses of samples taken from nominal 

### 221 mgll showedthatthe measured concentration was in agreement 

### with 

### what was anticipated and expected, i.e. 100 mgll (see Table 3 ofthe appended Analytical report). It should however be notedthat during exposure droplets were present on the. bottom ofthe exposure vessels. This could have had a direct effect on the immobility Ifthe droplets orlglnatetffrom undissolved pure test substance, which was expected based on the test substances specific gravity of , .47. Consequently, the observed immobility averaging 30% after 

### 24 hours and 80% after

### 48 hours should be taken with care. Furthennore, relatively high numbers of daphnlds were trappedatthe surface. Hence, the present test results were Influenced by mechanical disturbances and therefore considered nat representing the Intrinsictoxicity of the test substance. It was consequently decidedto continue with a ffnal flow-through studytesting a range of conoentratlons and to payspecIal 

### attentton not

### to Include undissolved 

### test substance droplets in the test vessels by increasing the stirring rate In the mixing vessels. The pH ranged between 

### 7.9 and 

### 8.0, the oxygen concentration was between 

### 8.4 and 

### 8.9 and Ihe temperaturewas kept between 

### 21.1 and 

### 21,SOC. Hence. all 

### test conditions in the limittest were maintained within the limits prescribed by the 

### protocol. Table 3 Incidence of immobility In the limittest

Concentration Vea.el Number Response at24 h Response

### at48 h (mgll) number Daphnia 

### exposed number Total % number Total % Blank-control A B C 0 5 5 5 5 0 0 0 0 0 0 0 0 O. 

### 0

100 A 8 C 0 5 5

! 5 5 0(5) 0(4) 5 1(2) 30 4 (2) 4 (1) 5 3(1) 80

Between brackets: runbet' of daphnlds observed trapped atthe swface. These Org&OI"'Swere relmmeraed intothe nrospectIve tdUllons before recording 01 mobility.
. 7.3. Final flow-through

### test 7.3.1. Measured concentrations The results of analysis of the samples taken during the final test are described in Table 4 of the appended Analytical Report and summarised in Table 4 on Ihe next page. Analyses of the samples taken from nominal 22 and 221 mg/l one day before the start of the test showed mean measured concentrations 

### of7.7 and 95 mgll indicating that the system functioned properly and that the actual concentrations were close to the antlclpated targets of 10 and 100 mgll. All concentrations measured atthe start ofthe experiment were In agreement 

### with what was expected, exoept atthe nominal concentration of 71 mgll. This concentration was lowerthan measured at nominal 40 mg/l. Concentrations remained stable at the two highest concemratlons, butdecreased In the lower concentrations. However, only the highest exposure concentration 

## was essential fordetermination ofthe toxicity parameters. The mean concentration measured In the highest test group corresponded to 94 mgll.


### -Page13­Table 4 Mean measured concentrationsversus nominal concet,tratlon Mean M

..sured Measured 

# I Concentration (mgn) m_sured t=O h t:::48 h (mgll) (mg/I) (mgll) Nominal I Expected' 4.7 1.6 2 10 7.8 22 15 20.4 10.4 40 18 8.4 12 71 

# Ht 32 16.3 78.3/36.73 51.7 123 55 104 94 88.2 221 j Based on the expected loss 01 testsuIlB1ance from waterdue to 8Y8pOIlltion. I Based on one meaau11IITlant aIndividual values gillen duetothehigh dlfler9l'lC8 In dupl'mtemeuu11IITl9nts 

### 7.3.2. Immobility Table 5 shows 

### the responses recorded during**th~** final flow-through test Immobility percentages ranged between 5 and 30% In the different concentrations tested. Hence, a 50% Immobility rate was not reached In 

### anyofthe concentrations tested. Including a highest tested concentration of 94 mglt. There was no clear relation between the actual exposure concentrations and the observed Immobility. Attermination ofthe studysome droplets were observed in the four highest exposure concentrations indicating a possible disturbing effecton the mobility rates 

### dueto undissolved test substance. Comparison of the results ofthe highest exposure concentration 

### with those obtained in 

### the limittest showed thatconcentrations were comparable but effects were significantly lower 

### in the final 

### test. Itcan be argued that the higher effects In the 

### limit.test were likelyto be related tothe presence of relatively large amounts of preclpftate (droplets) and the high numbers 

### offloaters. Floaters were not observed in 

### thefinal test. TableS Acute Immobillsationofdaphniaafter24 and 48 hours inthe final

### test

Concentration -(man) Vessel number Number Daphnia exposed Respon

.. at24 h Respon
.. at48 h number Total % number Total % Blank-control A 5 0 0 0 5 B 5 0 0 C 5 0 0 0 5 0 1 4.7 A 5 1 10 1 10 B 5 0 0 C 5 1 1 0 5 0 0 15 A 5 0 0 

### 0 5 B 

### 5 0 1 C 5 0 0 0 5 0 

### a 12 A 5 0 0 1 15 B 5 0 0 C 5 , 1 1 0 5 1 1 55 A 5 1 20 1 30 B 5 3 3 (1) 

## c 5 0 0 0 5 0 2 94 A B C 0 5 5 5 5 1 

### 0 0 1 10 1 1 (1) 0 3 25

! Between bn:lckeIB: number 01 daptJnlda obseMld trappedatthe surlaoe. These organIsms were reimmelse<f Into (he respective soIUIIons before rllCOfdtlg 01 mobBfty.

- Page 14­7.3.3. Determination ofefl'ectconcentrations Table 6 

### shows the effectparameters basedon target concentrations, 

### see also Appendix I. Table 6 Effect parameters Pal'llm8ter Concentration (mgll) NOEC Not determlnod 24h-ECso >94 48h·ECso >94
7.3.4. Experimental conditione The results of measurementof pH and oxygen concentrations (mg/l) 

### are presented in Table 7. These test conditions remained within the limits prescribed by

### the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: >7 mg/1 at the start,**~5** mg/1 atthe end 

### ofthe test). Thetemperature of the test medium 

### was 2O.5°C at the start of the test. The temperature continuously measured In a temperature control vessel varied between 20.3 and 20.7°C during thetest, and complied with the requirements as laid down in the protocol (18-22DC, constant within 

### 2OC). Table7 pH and oxygen concentrations duringthefinal test

Concentration (mal'll Start pH bOh) 02 End (1:=24 h) EndCh=48 h) 

### pH**~** pH 

### 02 Blank-control 8.1 8.8 8.1 8.8 8.1 9.0

4.7 8.1 8.7 8.1 8.9 8.1 8.9 15 8.0 8.7 8.1 8.8 8.0 8.8 12 8.0 8.6 8.1 8.S 8.0 8.9 55 7.9 8.6 7.9 8.4 8.0 8.7 94 7.9 B.6 7.9 8.3 7.9 8.6
8. CONCLUSION The4Sh-ECso exceeded a mean measured concentration of 94 mg/l undertheconditions of the present flow-through studywith Daphnia magna exposed to

- Page 15­IAPPENDIX 1 REFERENCE TEST Start: 

### 21 June

### 2005 End : 23June

### 2005 48-hour Acute ToxicIty Study In Daphnia magna with K2Crl!07 The study procedures described in this report were based on the ISO Intemationel Standard 8341, the EEC directive 92169, Part C.2. "Acutetoxicityfor Daphnia", December 1992, and 

### the OECD guideline 

### No. 202: "Daphnia sp., Acute Immobl1isation Test", Adopted April 

### 4. 1984. The reference test was carried 

### out to check

### the sensitivity 

### ofthe test system as used 

### by Daphnia were exposed for a maximum 

### of 48 hours to**KeCr2~concentrations** of 

### 0.10,0.18,0.32
. 0.56, 1.0 and 

### 1.8 mgll and toa blank control. Twenty daphnia were exposed perconcanlration. The reference substance, potassium dichromate 

### O<2CrzO" art. 4864, 

### batch no. K28974764) 

### was obtained from Merck, Darmstadt, Germany. Acute Immobilization ofdaphnia after

### 24 and

### 48 hours In th9 reference 

### test withporasslum 

### dichromate:

Concentration (mgn) Number Exposed % Immobile 24h 

### 48h ExpecIed 

### respon•• (%) After48 hoUrs ' Minimal Maxfmal Blank-eontrol 

### 20 0 0 0 102 0.10 20 0 0 0 10 0.18 20 

### 0 0 0 10 0.32 20 

### 10 5 0 30 0.56 20 

### 0 45 

### 0 100 1.0 20 80 I 

### 100 40 

### 100 1.8 20 

### 100 100 100 

### 100 Based on hIStorical data ofthe pl'e'lllOUS years (0)00). A maxhlum 1'8$pOI'I8&of 10% does not InYBIldalethe n:lSUlt& ofthetest The actual responses in this reference test with 

### KeCr207 were within the ranges 

### ofthe expected • responses at the different concentralions. Hence, the sensitivity of this batch 

### of Daphnia magna was in agreement 

### with the historical data collected at The 24h-ECsa was 0.84 mgt! with a 95% confidence Interval between 0.78 and 0.94 mgll. The 48h-EC50was 0.53 mgIJ with a 95% confidence interval between 0.46 and 0.64 mgll. The raw data from this study are f<ept in the archives. The fest described above was performed under 

### GlP with a QA-check.

• Page 16­

### APPENDIX II ANALYSIS OF TAP WATER 

### (Not performed under 

### GLP) 

### Date 

### of sampling : 08-06-05 

### Sample numbers :2106017 

### (kamer 25) Laboratory jCOMPONENT: Metals: Selenium Arsenic Cadmium Chromium Copper Mercury Lead ZInc Manganese· CalCium Magnesium Iron EOX (extractable organic halogens) PolycyclicAromat1c Hydrocarbons:

. Naphtalene

Acenaphtylene Acenaphtene Auorene Phenanthrene Anthrac::ene Auoranthane Pyrena Benzo(a)antraoene Chrysene Benzo(b)ftuoranthene Benzo(k)ftuoranthene Benzo(a)pyrene Dibenzo(ah)antracene Benzo(ghl)perylene Indeno(123cd)pyrene
PAC'sTotal EPA (18) PAC'sTotal VROM (10) ANALYSIS:

<0.90 <5.0 <0.40 <1.0 

### 29 <0.050 <5.0 19 <0.010 53

5.2 <0.050 <1.0
<0.010 <0.050 <0.010 <0.010 <0.010 <0.0050 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 ..gil 

### pgll

..gil 

### pgll., pgll pg/l 

### pgn IJg/I

## '~I)' mglll) mgI1 IJg/I pgII 

### pgl1 pgl1 IJg/I pgI1 pgI1 

### pgl1 pgll JIgl1 pgl1 J.Ig/I pgI1 IIQI! J.Ig/I J.Ig/I 

### IJVIl

Total aerobic gerrn-oount {22VC} 2 CFU/ml 1lJ Total aerobic germ-count {37!IC} 2 CFUlml1) Hardness: 1.6 mmoll1a) Nitrate (expr. as N); 0.67 mg/T Nitrite (expr. as N); 0.018 

### mgn Cyanlde-Iotasl (NEN 6655) <1.0 ugli Turbidity 0.049 

### FTE Colour Intensity 20 mgPt/1 Dechlorination of

### tap water was not necessary since no chlorine 

### was present. 'Tap water was sampled from 1118copper-free wat\M supply system1I1atsupplied theWIlIer used for the presenttoxlclly study. Amongo1h$l8, oopperand baclBrlal activity

__maMCnd intheae samples. The alher comPOrnlflta

__ analysed InII8/TIPkIG taken from 1hesame waI8rsupplybutsampled In ttle Animal House

.

• Page 17­

## P-04-635 

## APPENDIX III

ACUTE TOXICITY STUDY IN 

## DAPHNIA MAGNA WITH (FLOW-THROUGH); DETERMINATION OF THE CONCENTRATIONS

Study

### completion date September 13, 2005

laboratory ProJect Identification

- Page 1 of 15 •1. CONTENTS



# . 1. CONTENTS

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

### 2**2~** REPORT APPROVAL

............................................................................................................3 

### 3. INTRODUCTION

....................................................................................................................4 3.1. Preface

...........................................................................................................................4 3.2. 

### Aim ofthe study

..............................................................................................................4 4. MATERIALS AND METHODS

...............................................................................................4 4.1. Reagents

........................................................................................................................4 4.2. Samples

..........................................................................................................................4 4.3. Analysis

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

### 5 4.3.1. Analytical method

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

### 5 4.3.2. calibration

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

### 5 4.3.3. Injections

..............................................................................................................5 4.3.4. Data acquisition and data processing

..................................................................5 4.3.5. Procedural recovery samples

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

### 6 4.4. Interpretation

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

### 6 4.4.1. Calculations

....................................................................;.....................................6 4.4.2. Formulas

..................................................................................:...........................6 5. RESULTS

...............................................................................................................................8 

### 5.1. calibration curves...........................................................................................................

### 8 

### 52. Samples

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

### 8 5.2.1. Procedural recovery samples

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

### 8 

### 5.2.2. Testsamples

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

### 8 TABLES Table 

### 1 Procedural recovery samples

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

## 9 Table 2 Concentrations of In test medium (range-finding test)

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

### 11 Table 

### 3 Concentrations 

### of in test medium (lIow·through limit test)

........ 11 Table 4 Concentrations of 

### In test medium (final flow-through 

### test)
........ 12
FIGURES Figure 1 GC chromatograms of 

### a 0.0780 mgll calibration solution [top; res.id. 3139] and blank methanol [bottom; res.ld. 3150]

.............................................. 13 Figure 

### 2 GC chromatograms 

### ofprocedural recovery samples 

### at concentrations 

### of0.0975 Figure 

### 3 GC chrometograms of blank control samples taken at 

### t=O hours [top; res.ld. 2884] Figure 4 GC chromatograms 

### oftarget 10 mglJ samples taken at t=-24 hours [top; res.ld. Figure 5 GC chromatograms 

### oftarget 100 mgll samples taken att
....24 hours [top; res.ld. mgll [top; res.id. 3154) and 97.5 mglllboHom; res.ld. 3155]

........................................ 13 and 

### att=48 hours [bottom; res.ld. 3234]

...................................................................... 14 

### 2534J. at t=O hours [middle; res.ld. 2885] and 

### att=48 hours [bottom; res.ld. 3235J

.... 14 

### 2536J. at 

### t=O hours [middle; res.ld. 2892] 

### and at t=48 hours 

### [bottom; res.ld. 

### 32281

.... 

### 15

- Page 2 ­

### 2. REPORT APPROVAL
PRINCIPAl SCIENTIST: {Analytical Chemistry)

--:::.......................................

Date:

## ,.~; '1.£A)';­

-Page3­3. INTRODUCTION 

### 3.1. 

### Prmce Study plan (analytical study) Start Completion : 30 May 2005 : 26July2005 

### 3.2. Aim oftheltudy The purposeofthe analytical study was to determine the actual concentrations in samples taken from thetest solutions usedduring the ecQtoxicily test. 

### 4. MATERIAlS AND METHODS 4.1. Reagents Methanol HPLC-grade. Labscan, Dublin,. Ireland MiJli-Q water Tap water purified by reversed osmosis and subsequently passed over activated carbon and ionexchange cartridges; 

### Millipore Corp, 

### Bedford. MAo USA ISQ.-medium 

### see main 

### report Tapwster see main report 

### 4.2. 

### Samples Ail samples not analysed on the sampling day were stored frozen. Stability ofthe samples under these conditions was determined during -Development and validation of an analytical method for the analysis of In MiIIi-Q water". On the dayof analysis. thefrozen samples were defrosted at room temperature. The samples (10 ml) were analysed using headspace (see 4.3.1 analytical method).



### -Page4- i j 

# i •4.3. AnalysIs 4.3.1. Analytical method Quantitative analysis was based on thetest substance peak In the GC chromatogram of 

### Analyflca/condNlans Column Stationery phase Dimensions Brand Temperature program Initial temperature Inilialtime 

### Rate 1 Temperature 1 Rate 2 Final temperature Hold 

### time Carriergas Column flow Injection Temperature Splitless time Injection volume Injection speed Headspace conditions Sample Incubation temperature Incubation time Syringetemperature Detection 4.3.2. 

### Calibration CP Porabond Q 25 m 

### x 0.25 mm, df= 3 I-Im Varian, Mulgrave, 

### VIctoria, Australia
40"C 1 min 20°Clrnin 190"C 50 GC/min 

### 250 ·C 3.3 min Helium 1 mVmln Splitless 

### 250 

## "c 2 minutes 500 IJI (Headspace) 50 IJIIsec
10 ml

### ofan aqueous sample in a 20 mlvial SO"C 

### 30 minutes 110°C FlO (250 GC; Hydrogen: 40 mllmin; Air: 450 ml/min)
During the range finding test, calibration solutions in ISO-medium

### were made up from two standard solutions of in methanol. During the flow-through limitand final flow-through

## test. calibration solutions In 

### tap weter were made up from two standard solutions 

### of In methanol. 4.3.3. Injections Calibration solutions were Injected In duplicate. Procedural recovery samplesand test samples were analysed by single Injection. 4.3.4. Data acquisition and data processing Data acqUisition and data processing was perfonned using Empower software (Waters. Milford, MA, USA)version 5.00.
·Page5­4.3.5. Procedural recovery samples Standard solutions of in methanol and dilutions ofthese solutJons with methanol were used asspiking solutions. During the range-finding test, ISO-medium (10 ml)was spiked atconcentration levels of 0.101 mg/l and 101 mgll using 10.1 mg/l and 10085 mgll spiking solutions 

### In methanol. During the flow-through limit test on 28-06-05 and 29-06-05. tap water (10 ml) was spiked at concentration levels of0.101' mgll and 101 ' mgll using 10.1 mg/l and 10150 mg/l spiking solutions 

### in methanol. During the flow-through limittest

### on 04-07-05, tap water (10 ml)was spiked atooncantration levels 010.101' mgII and 101' mgll using 10.1 mg/l and 10119mgll spiking solutions In methanol. During the final flow-through tastfrom 19·07-05 and 20-07-05, tap water(1 0 ml) was spiked 

### at concentration levels of0:100' mgll and 97.4' mgll using 10.0 mglt and 9740 mgll spiking solutions 

### in methanol. During the final flow-through test on 25-07-05, 

### tapwater (10 ml)was spiked at concentration levels of0.0975' 

### mgII and 97.5' mgJl using 9.75 mg/l and 9746 mg/l spiking solutions In methanol. At approximately0.1 mg/l one and at 100 mg/l 

### five sampleswere prepared 

### and treated as described In paragraph 4.2 'Samples' and analysed. The recoverywas calculated 

### for each semple 4.4. 

### Interpret.Uon 4.4.1. calculations 

### All calculations were performed using non-rounded values. Rounded values are reported in the 

### tables. Therefore, some differences mightbe observed when calculating the parameters as mentioned In the tables. 4.4.2. Fonnulas**~eneral**



### _ 1n Mean: 

## x=-I: XI 

### n 1-1 where :xi 

### = measured 

### value 

### n :: numberofmeasurements 

## " L(Xi-X}2 Standard deviation Sn _I =**U-,=I=~l** --,-­ (n-l)
Coefficient ofvariation 9n,:I. 

### 1oo% X

I Concentrationswere not corrected for spiking volume

- Page 6 •Calibration Response: R 

## = Peak area test substance [units] Calibration curve: The responsewas correlated 

### with the test substance concentration, using linear regression analysis (least squares method; weighting factor (1!concentration2)).

R 

# = response calibration solution [units] 

### C 

# = concentration of

### test substance in calibration solution [mg/ll a 

### = slope [unlts.Umg] b 

# = Intercept [units]**~amples** Concentration ofASAHIKLIN AE-3000 analysed In the samples:.

R 

### = response sample [units) d 

### = dUunon factor 

### a ::z slope {unlts.Vmgl b 

### = intercept [units) Recovery of procedural recovery samples: Concentration analysed • 

### 100 [%] Concentration 

### prepared Concentration analysed • 100 [%] Relative 

### to nominal concentration: Concentration nominal Relative to mean initial concentration: Concentration analysed at 

### t 

## =x hours • 100 

### (%] Mean concentration analysed at 

## t= 0 hours Concentration analysed II< 

### 100 [%) Relative to target: Concentration target



### - Page 7­ 

## i 

# I 

# I

### 5. RESULTS 

### 5.1. Calibration curves On each day ofanalysis. acalibration curve 

### was constructed using six concentration leve!s. At each concentration level, two responses were used. The coefficient ofcorrelation was in excess of0.99 

### for 

### each calibration curve. Agure 1shows chromatograms of

### a calibration solution and blank methanol. 

### 5.2. 

### Sample. 5.2.1. Procedural 

### recovery samples Figure 2 shows chromatograms of procedural recovery samples. The results for the procedural recovery sampfes are summarized In Table 1. Mean recoveries of procedural recovery samples are required 

### to be In the range 

### 70-110%. Because the recoveries ofthe procedural recovery samples were met, the results 

### ofthe test samples 

### ware considered accurate. 

### 5.2.2. Test

### samples Figures 

### 3 - 5 show chromatograms 

### ofblank control samples, nominal 

### 1 0 mgn samples, and nominal 

### 100 mg/l samples. The results for 

### the test samples 

### are summarized in Tables 

### 2 - 4. During the flow-through tests, a small peak at the retention time ofthe test substance was observed in the chromatograms 

### ofseveral blanktest samples (nominal 

### 0 mgll). Because a similar peakwas found In blanks 

### ofmethanol Itwas concluded that the compound did not derive from test medium. For most ofthe blank test samples, 

### the response ofthe interference was smaller than fhe response ofthe lowest calibration solution, therefore < LOQ was given. For one blank test sample a response within the concentration range ofthe calibratIon solutions 

### was found and therefore an accurate value was given. 

### The maximum contribution ofthe compound to the peak area ofthe lowest test sample was 

### 0.6%, and was therefore notconsidered 

### to be a significant interference.



### ·PageS­

### TABLES Table 1 

### Procedural recovery samples Recovery Concentration Mean recovery 

# I (Coefficient of Concentration Date of Date of 

### analysed nominal 

## r.41 analysis 

### preparation variation) : [mgll] [mgll] [dd.mm-yy] [dd-mm-yy]

# ~I 108 0.109 0.101 30-05-05 1 

### 30-05-05 30-05-05 1 

### 98 101 98.6 101 30-05-05 78 101 79.1 91 101 104 

### 105 (15) 101 99 

### 100 74 108 74.2 0.109 28-06-05 1 

### 0.101 28-06·05 28-06-05 1 

### 98 101 99.7 101 28-06-05 98 

### 99.4 

### 99 101 98 

### 99.0 101 (1.3) 101 101 102 99 

### 100 0.0813 

### z 80 

### , 0.101 29-06-05 29-06-05 ' 

### 96 101 97.6 101 29-06-05 29-06-05 1 

### 95 101 96.7 

### 95 101 96.3 95 (1.3) 101 93 94.8 93 0.0989 2



### 94.8 98 04-07-05 1 

### 0.101 04-07-05 04-07-05 1 

### 86.0 85 

### 101 101 04-07-05 

### 92 101 92.8 87.7 90 

### 87 (4.1) 101 91.4 90 

### 101 94 

### 19-07-05 95.0 104 19-07-05 0.104 0.100 19-07-05 104 101 97.4 19-07-05 90 87.5 97.4 101 104 97.4 101 106 97.4 104 

### (6.5) 101 98.6 97.4

-Page9­Table 1 

### Continued Concentration Concentration Date of Date of Recovery 

# ! Mean recovery nominal analysed analysis 

### [%J i (Coefficient of [dd-mm-yy] preparation [mgll] [mgll] variation) [dd-mm-yy] 

### r%l 0.109 109 20-07-05 0.100 20-07-05 97.4 83.7 86 20-07-05 20-07-05 97.4 87.2 89 97.4 

### 88.2 91 89 

### , 97.4 85.9 88 

### (2.0) 97.4 87.1 89 0.0975 0.0866 25-07-05 25-07-05 89 95.9 25-07-05 97.5 25-07-05 98 97.5 98.4 101 97.5 100 

### 101 103 99.5 97.5 

### 102 

### (2.0) 96.3 99 97.5 Combined with ConcenlraHon was below\he Umltofquanllflcation. butwithIn the concen1ration range ofthe ealibration solUuons.

-Page 10­Table 

### 2 Concentrations of In test medium (range-finding test) 

### I Tlmeof • sampling [hours] Date of sampling [dd-mm-yyJ Date of analysis [dd-mm-yy] 1 Concentration Nominal

[mgl\] Analysed [mgIJ] Relative to nominal 

### [%1 Relative to mean Initial 

### r%l 0
48 23-05-05
25-05-05 30-05-05
30-05-05 100 2 100 2 

### 100 100 3.71 

### 6.58 3.32 6.86 4 7 

### 3 7 65 

### 133 samples were frozen unlll analysis. Combined with

Table

### 3 Concentrations of in

### test medium (flow-through limittest)

1 Concentration 

## i Time of Date 

### of Date 

### of Relative to sampling analysis sampling Target Analysed target [dd-mm-yy] [dd-mm-yy] [hours] [mgl\] [mgl\] f%] 

## I 28-06-05 93.7 -24 28-06-05 221 42 221 

### 102 46 

### I 29-06-05 29-06-05 

### 0 n.d. 

### 0 n.a. 

### 0 

### n.d. n.8. 

# . 221 95.7 43 221 100 

### 45 <LOQ2 01-07-05 04-07-051 

### 48 0 n.a. <LOQ2 

### 0 n.a. 221 

### 98.5 45 221 99.7 45 

# I Samples werefrozen un1I ana1y.si5. LOQ 2 A small peakwas obseM!d atthe retenllon limeoflestlIub11ance, butIts response was not ccnsidered to derive from test sample since itwas also obserwd In blank m9lt1aoo1. Umitofquantification 'ME 0.100 mgll (See "Development andvalidation ofan analytical method for It. analysis of In MIIII-Q**waler"~** n.d. Not detected n.8. Notapplicable

- Page 11 ­in test medium (final flow-through test) Table 4 Concentrations of Concentration

Time 

### of sampling

[hours] Date of sampling [dd-mm-yy] Date of analysis [dd-mm-yy] Target 

### [mgll] Analysed [mgn] Relative to target



### f%1 -24 

### 19·.Q7-05 ,19-07-05 22 7.80 35 

### 22 7.64 35 221 96.8 44 

### 221 93.7 42 0 20-07-05 20-07-05 0 <LOQ2 

### n.a. 0 0.130 

### n.a. 22 7.60 35 

### 22 8.00 36 

### 40 20.3 51 

### 40 20.5 51 71 15.3 22 

### 71 15.3 22 123 54.0 44 

### 123 49.3 40 221 90.2 41 221 86.2 39 48 

### 22-07-05 26-07-051 

### 0 0 <Loa2 <lOO2 n.a. 

### 22 1.56 7 22 

### n.1. n.a. 

### 40 10.4 26 40 10.4 26 71 8.55 12 71 8.20 12 123 78.3 :5 

### 64
, 123 36.7 3 

### 30 221 105 48 221 102 46 Slwnples ware frozen unb1 analysla, 

### Loa Z 

### Asma. peak was observed at the I'8tentlon !meoflestsubstance. but Its response W8B not considered to <IerIYe from test sample sir1C8 itwas also obselYBd in blank methanol. LImit ofquanllf\caUonwas 0.100 mgll (See :"Dllwloprnentand validatioo ofan anCllytlca1 method lorthe analysis of In MlnJ·Q wate!"). No analyUcaI explanaUon can be given forthe dlfflll'8nflal results ofthe duplicate samples. n.a. Notapplicable n.l. Not

### iJVectvC (sample viCiI we8 broken).



### - Page 12­1 FIGURES



# - Figure 1 GC chromatograms ofa 0.0780 mgll calibration solution 

### [top;
... Id. 3139} and blank methanol [bottom: resold. 3150].

1.



# ..: 

# .. ..... Figure 2 GC chromatograms of proceduraJ recovery sample. atconcentrations of 0.0975 

### mgtl [top; res.id. 3154] and 97.5 

### mglllbottom; res.ld. 3155].

# _. "

# .. Figure 

### 3 GC chromatograms ofblank control samplestaken att=O hours 

### [top; res.ld. 2884} and att=48 hours [bottom; res.ld. 3234]
.



# .

# ~.~---~~ ...

# ~... ­

# .~**-~--.~****·~*----1~-----~----.-&----.~----_-i--~_-i-----~~****~ .-~----~-_----H!.----Q-~----1~** 

- Figure 4 GC chromatograms oftarget 10 mgll samples taken at f:lL.24 hours 

### [top; res.ld. 2534]. att-o hours [middle; resold. 2885] and att=48 hours [bottom; res.ld. 3235J.

# .~,oJ---~~

# .~~------------~.~, -----­

# .:j~~~--~~~--.~-=~~e---~~~~:~==~.~,--~~~,..

# ~&~~~.~-=~~.~~..

# ~.~~_~~.~ - Figure 

### 5 GC chromatograms of

### target 100 mgllsamplestaken 

### at t=-24 hours [top; 

### res.ld. 2536), 

### att=O hours 

### [middle; res.ld. 2892) and att=48 hours [bottom;

..... Id. 3228].

- Page 15­

## P-04·635

LOCAL L¥MPHNODE ASSAY REGULATORY/REPORT
DATA REQUIREMENT: EPA OPPTS 870.2600 (2003) OECD 429 (2002)
AUTHORS:
STUDY COMPLETION DATE: 27 October

### 2005
PERFORMING LABORATORY:

LADORATORYPROJECT ID: Study Number: Report Number: Contract Number:


### SPONSOR:

Report Number: 

### Page 1of21**GOODLABORATORYPRACTICECO~LMNCESTATEMENT** 

### I, the undersigned, declare that the objectives laid down in the protocol were achieved and 

### that the data generated are valid. Thereport fully and accuratelyreflects the procedures used and the raw data generated in the above study. The study (GM7907)was conducted in compliancewith the UK Principles ofGood Laboratory Practice (The United Kingdom GLPRegulations 1999, StatutoryInstrument No. 3106, as amended 2004, StatutoryInstrument No. 994) except for thedeviation listedbelow. These Principles 

### are inaccordance 

### with the OECD PrinciplesofGood LaboratoryPractice, revised 1997 (ENVlMClCHEM(98)1

### 7).
Thefollowing GLP deviation is considered not to affect the integrityofthe studyorthevalidity oftheconclusions drawn: 

### (i) the stability and achieyed concentration ofthe test substance in the vehicle used were not determined by analysis.


### 27 October

### 2005 StudyDirector Date Research and Investigative Toxicology Performing Laboratory:

ReportNumber: Page 2 of 21

### Report Number: 

### Page 3 of 21

### QUALITY

### ASSURANCE STATEMENT In

### accordance ",rith 

### policy and QA 

### procedw-es for Good Laboratory Practice, this report has been audited and the conduct ofthis study has been inspected as follows:

Date AuditlInspection Date ofQA

### Report 04 Aug 2005 

### Draftreport 09 Aug 2005 22 Oct 2005 Final reportreview 22 

### Oct 2005

In 

### addition, inspections associated with this type ofstudy were made as follows: 04 Apr

### 2005 Dose preparation 04 Apr2005 20 Apr 2005 

### Dose administration, bodyweights 22 Apr2005 16 

### May 2005 Protocol 16 

### May 2005 23 

### May2005 Radiolabelled preparation, injection of 23 

### May 2005 radiolabel 23 

### May 2005 Processing, removal ofJymph nodes 24 May 2005 07 Jun 2005 Scintillation counting 14Jun2005

Facilities and process based procedures associated with 

### this type

### ofstudywere inspected 

### in accordance with 

### QA

### Standard Operating Procedures. So far as can bereasonablyestablished, the methods described and theresults given in the final report accuratelyreflect the raw data produced during the study, GM7907.

27 October2005 Quality Assurance Unit)

Report Number: Page 3 of 19


### ReportNumber: 

### Page 4 of 21

### Report Number: 

### Page 5 of 21

## GENERAL INFORMATION

Contributors The following contributed to this report in thecapacities indicated: Name Title Study Director Study Licensee Study Reviewer


### Studydates Study initiation date: 171h 

### June 2005 Experimental start date: 22nd 

### June 2005 Experimental termination date: 281h 

### June 2005 

### Deviations from the guidelines None 

### Retention of

### samples Anoriginal report, the study protocol and all rawdata, samples and specimens pertaining to this study are 

### retained in the Archives,
. for a minimum of5

### years from the date ofissue ofthe final report. At the end ofthis period the Sponsor will becontacted regarding thefurther retention, return to the Sponsoror destruction ofthe archived materials.

Report Number: Page 6 

### of 21

## TABLE OF

## CONTENTS

THE TITLE PAGE 

### 1 GOODLADORATORY PRACTICE COMPLIANCE STATEMENT defined. QUALITYASSURANCE STATEMENT GENERAL INFORMATION 

### 6 TABLE OFCONTENTS 7 

### 1.0 EXECUTIVE SUMMARY 

### 9 1.1 Study design' 

### 9 1.2 Reswts 

### 9 1.3 Conclusion 

### 9 

### 2.0 INTRODUCTION 

### 10 2.1 Purpose 10 2.2 Regulatory guidelines 10 2.3 Justification for test system selection 10 2.4 Positivecontrol study 10 2.5 Study dates 10 2.6 Data storage 10 

### 3.0 MATERIALS AND METHODS 

### 11 3.1 Test substance 

### 11 3.2 Positivecontrol substance 11 3.3 Vehicles 11 3.4 Dose preparations 11 3.5 Experimental design 12 3.5.1 Animals 12 3.5.2 Accommodation andhusbandry 12 3.5.3 Acclimatisation 12 3.5.4 Animal identification 13 3.5.5 Test method for theevaluation of 13 3.5.6 Clinical observations 13 3.5.7 Bodyweights 13 3.5.8 Positivecontrol study 13 3.6 Dataevaluation 14

ReportNumber: Page 7 of 21

### 4.0 RESULTS 14 4.1 Evaluation oftest substance (Table 1) 

### 14 4.2 Positive control (Table

### 2) 14 

### 5.0 CONCLUSIONS 14 

### 6.0 REFERENCES 15 

### TABLES SECTION 16 TABLE 1 SKIN SENSITISA TION POTENTIAL OF 17 

### TABLE 2 SKIN SENSITISA TION POTENTIAL OF THE POSITIVE CONTROL SUBSTANCE (HEXYLCINNAMALDEHYDE) 

### 18 

### APPENDICES SECTION 19 APPENDIX 1 BODYWEIGHTS 

### (g) 

### 20

Report Number: Page 8 or'21

## 1.0 EXECUTIVE SUMMARY 1.1 Study design A sample of was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay. Theassay determines the level ofTlymphocyte proliferation in the lymph nodes draining the site ofchemical application, by 

### measuring the ammmt ofradiolabelled thymidine incorporated into the dividing cells. The test substance was applied as 25, 50 or 100% 

### w/v preparations in acetone. A 

### vehicle control group was similarly treated using acetone alone. 

## 1.2 Results

• Thetest substance did nothavethe capacity to cause skin sensitisation when applied as 25 or50 

### %w/v preparations in acetone orwhen applied neat. In a positive control study, hexylcinnamaldehyde was shown to havethe capacity to cause skin sensitisation when applied as 10% and 25% 

### w/v preparations in acetone in olive oil (4: 1), 

### confirming the validity ofthe protocol used for this study. 

## 1.3 Conclusion In conclusion, is unlikely to be a skin sensitiserunderthe conditions ofthe test.

ReportNumber: Page 9 

### of 21

## 2.0 INTRODUCTION 2.1 Purpose Thepurpose ofthis study was to assess the skin sensitisalion potential of using the Local LymphNode Assay (Kimber and Basketter 1992, Kimber etaI19Y4). 

## 2.2 Regulatory guidelines This study was conducted in accordance with the following Regulatory Guidelines: a) OECD guideline reference 429 (2002): Skin sensitisation: Local Lymph Node Assay. b) United States Environmental Protection Agency, Health Effects Test Guidelines, OPPTS 870.2600 (2003): Skin Sensitisation. 

## 2.3 Justification for test system selection The CBAlCamouse is the species and strain ofchoice because previous examination ofstrain difference inlymphocyte proliferation responses to skin sensitisers showed this strain ofmouse to exhibit the most vigorous response (Kimber etai, 1994). 

## 2.4 Positive control study The reliability ofthetest system is assessed at approximately 6-monthly intervals using a known sensitiser (hexy1cinnamaldehyde, Dearman etai, 

### 2001). The positive control study closest in time to the main study is included in this report. 

## 2.5 Study dates The main study was initiated on 17th 

### June 2005. The experimental phase started on 22nd 

### June 2005 and 

### was completed on 28th 

### June 2005. Forthepositive control study, the experimental phase started on 15th 

### June 2005 and was completed on 21 st

### June 2005. 

## 2.6 Datastorage An 

### original report, the study protocol and all raw data, samples and specimens pertaining to this study are retained in the Archives, fQr a minimum of5 years from the dateofissue ofthe final report. At the end ofthis period the Sponsor

### will be contacted regarding the further retention, return to the sponsor ordestruction ofthe archived materials.

Report Number: Page 10 of 21

## 3.0 MATERIALS AND METHODS 3.1 Test substance

Name: Source: Colour: Colourless Physical state: Liquid Batch referencenumber: test substance reference number: Purity 

### (% w/w): 99.99% 

### Storage conditions: Ambient temperature in the dark Stability: 

### 21th September 

### 2005 From information supplied by 

### the Sponsorthe test substance was used within the expiry date. A certificate ofanalysis (dated 

### 21th September 

### 2005) is retained in the Archives. The test substance characterisation was the responsibility ofthe Sponsor.


### 3.2 Positive control substance

Name: Hexylcinnamaldehyde Source: Aldrich Chemical Company Colour: Yellow Physical state: Liquid test substance reference,number: Storage conditions: Refrigerated, under an inert gas, in the dark' 

### 3.3 Vehicles The vehicle for the test substance was acetone (I 

### The vehicle for the positive control substance was acetone in olive oil (4: 1;


### 3.4 Dose preparations All dose preparations were used within 24 hours ofpreparation.

ReportNumber: Page 11 of 21

### No correction was made for the purity ofthe active ingredientin the test substance. Stability and achieved concentration were not determined. 

### 3.5 Experimental design 3.5.1 Animals

Species: Strain: Source: Sex: Number used: Specification: Mouse CBAlCa Charles River Female 4 per group Young adults (8-12 

### weeks ofage) 

### 3.5.2 Accommodation and husbandry A 

### maximumof4 

### mice was housed percage, in cages suitable for animals ofthis strain and weight range. Environmental enrichment provided included tents, bases and nestlets. The animal room was designed to give the environmental conditions shown as follows. Temperature: 22±3°C 

### Relative humidity: 30-70% 

### Air: A minimum of 15 changes perhour Light cycle: Artificial, giving 12 

### hours light, 12

### hours dark Both temperature and relative humidity were recorded daily. In

### general, the recorded values were within the specified ranges and any deviation is considered not to affect the integrity ofthe study. Diet , supplied 

### by and mains water, supplied by an automatic system, were availableadlibitum. Each batch ofdiet is routinely analysed for composition and for contaminants. Wateris also periodically analysed for contaminants. No contaminants were found in the diet or waterat levels considered likely to interfere with the purpose or outcome ofthe study. Certificates of analyses areretained in the 

### 3.5.3 Acdimatisation The animals were housed under the experimental conditions for atleast 5 days, prior to**th~** 

### start ofdosing.

ReportNumber: Page 12 

### of 21

### 3.5.4 Animal identification 

### Animals were individually identified, within each cage, by aclipped areaon the side(s). The first animal 

### in each cage had a clipped areaon the left side, the second on the righthand side, the third on both flanks and the fourth not clipped. 3.5.5 Testmethod for theevaluation of 

### Groups offour female mice were used for this study. Approximately 25JlI ofa 25, 50 or 100% w/v preparation ofthe test substance in acetone was applied, using avariable volume micropipette, to the dorsal surface ofeach ear. Avehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days. Three days after the third application, all the animals were injected, viathe tail vein, with approximately 250JlI ofphosphate buffered saline (PBS) containing 20J.1Ci ofa 2.0Ci/mmol specific activity 3H-methyl thymidine. Approximately 5hours later, the animals were humanely killed by 

### inhalation ofhalothane vapourfollowed by 

### cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container ofPBS. A single cell suspension was prepared by mechanical disaggregation oflymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10rnl ofPBS. Approximately 3rnl of5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 rnl ofTCA. The lymph node suspensions were transferred to scintillation vials and 10rnl ofscintillant (Optiphase) was added priorto J3-scintillation counting using aPackard Tri-Carb 31OOTR 

### Liquid Scintillation Counter. 3.5.6 Clinical observations 

### Animals were checked at least once daily for signs ofsystemic toxicity. 3.5.7 Bodyweights 

### The bodyweight ofeach animal was recoded priorto dosing as day 1and prior to injection of3Hmethyl thymidine and day 6bodyweights are given in Appendix 1. 3.5.8 Positive control study 

### The sensitisation potential ofhexylcinnarnaldehyde was assessed using the method described above in 3.5.5.

ReportNumber: Page 13 of 21

### Approximately 25/l1 ofa 5%, 10% or25% w/v preparation ofhexylcinnamaldehyde in acetone in olive oil (4: I) was applied, and a vehicle control group was similarly treated using acetone in olive oil alone (4:1). 

### 3.6 Data evaluation Theresults are expressed as a disintegrations perminute (dpm) value perlymph node for each group. The activity ofeach test group is then divided 

### by the activity ofthe vehicle control group to givea testcontrol ratio known as the stimulation index (SI), for each concentration. The criterion for a positive response is that oneormore concentrations ofthe test substance should elicit a 3-fold orgreaterincreasein isotopeincorporation relative to thevehicle control group. The assay is able to identifY those materials thatelicit responses in standard guinea pig tests for skin sensitisation (Kimber etaI1994). 

### Consequently, a test substance which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser. 

## 4.0 RESULTS 4.1 Evaluation oftest substance (Table 1) The application ofthe test substanceat concentrations of25, 50 and 100% w/v in acetone resulted in an isotope incorporation which was less than 3-fold at all concentrations. Consequently, thetest substance is designated as unlikely to be a skin sensitiser underthe conditions ofthe test. 

### 4.2 Positivecontrol (Table2) The application ofhexylcinnamaldehyde 

### at concentrations of5%, 10%and 25%

### w/v in acetone in olive oil (4: 1) resulted in a greaterthan 3-fold increase in isotope incorporation atthe 10 and 

### 25 %w/v concentrations. Therefore, hexylcinnamaldehyde was shownto be a skin sensitiser, confirmingthe validity ofthe protocol used forthe study. 

## 5.0 CONCLUSIONS In conclusion, is unlikely to be a skin sensitiserunderthe conditions ofthe test.

Report Number: Page 14 of 21

## 6.0 REFERENCES Deannan R J, 

### WrightZ M, BasketterD A, Ryan C A, Gerberick G F and KimberI (2001), The suitability ofhexyl cinnamic aldehyde

## as a calibrant for the murinelocal lymph node assay. Contact Dermatitis, 

### 44, 357-361. Kimber I 

### and Basketter D A (1992). The murine local lymph node assay: a commentary on collaborative studies and new directions. Fd ChemToxic 30, 

### 165-169. KimberI, Deannan RJ, Scholes EW and BasketterDA (1994). The Local 

### LymphNode Assay: Developments and Applications. Toxicology, 

### 93, 13-31.

Report Number: Page 15 of 21

## TABLES SECTION

ReportNumber: 

### Page 16 of 21

### TABLEt SKIN SENSITISATION POTENTIAL OF
Concentration of test substance (% w/v) 

### Number oflymph nodes assayed Disintegrations per minute (dpm) dpmper lymph node Test: control ratio (SI) 

# o(vehicle only) 

### 25 50 100 8 

### 8 8
8 

### 3352 3748 3500 2813 419 469 438 352 N/A 

### 1.1 

### 1.0 0.8

Nt

### A - not applicable

ReportNumber: Page 17 of 21

## TABLE2 SKIN SENSITISATIONPOTENTIAL OFTHE POSITIVE CONTROL SUBSTANCE (HEXYLCINNAMALDEHYDE)
Current StudyNo.: 

### Concentration of hexylcinnamaldehyde (% w/v) 

### Number of lymph nodes assayed Disintegrations per minute (dpm) dpmperlymph node Testcontrol ratio (SI) 

# o(vehicle only) 5 10 25 8 8 8 

### 8 1636 3793 6821 24807 205 474 853 3101 N/A 

## • 2.3 4.2 15.1


### NI A - not applicable

Report Number: Page 

### 18 of 21

## APPENDICES SECTION

ReportNumber: 

### Page 19 of 21

## APPENDIX 1 BODYWEIGHTS (g)

DOS&: 0 %w/w
ANIMAL DAY DAY NOMBBR 1 6
If'I!!Ml\.LES 45 17.3 20.0 46 l1L4 21.7 47 lB.9 21.5 4B 19.B 22.4

DOSE: 25 "W/V
ANIMAL DAY DAY NOMBBR 1 6
1"I!lKII.I.ZS 451 15.B lB.4 50 19.2 21.1 51 151.7 22.4 52 lB.O lB.4



### Report Nwnber: Page 20 of 21

### APPENDIX 1 BODYWEIGHTS (g)

OOS8: 50 

# ""IV
ANlJ!oIIU. DAY DAY NUMII!.R 1 6
l!'DlIlUJIIS 53 lIL3 20.2 54 19.0 20.7 55 1S1.8 21.2 56 20.4 22.1

00511: 100 %W/W
ANIMAL DAY DAY NtlMB!.R 1 6
I'BMlUoII:S
57 18.5 19.5 58 18.3 19.7 sg 18.0 lSI.7 60 20.3 22.4



### Report Number: Page 21 of 21