# REPORT
StudyTitle: ACUTE TOXICITY OF DEA/ACID ANHYDRIDE REACTION PRODUCT TO ZEBRA FISH (BRACHYDAN/0 RER/0) IN A 96-HOUR SEMI-STATIC TEST
Data Requirements ITest Guidelines: OECD No. 203 EU Commission Directive 92/69/EEC, C.1 Study Director: Roger

**B~Hscher** Study Completion Date: November 19, 2004 Test Facility: RCC Ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen I Switzerland Sponsor: Rockwool InternationalAIS Hovedgaden 584 DK-2640 Hedehusene Denmark Identification: RCC Study Number: 848630 Page 1 of52RCC Study Number848630 DENAcid anhydride reaction product 

# GLP CERTIFICATE REPORT

The Swiss GLP Monitoring Authorities

Swiss Federal Officeof Public Health SwissAgencyforthe Environment, Forests and Landscape ./ Swissmedic

**~** • SwissAgencyfor 

## SW1ssrned1c TherapeullcProducts 

# Statement of GLP Compliance

It is hereby confirmed that during the period of thefollowingTestFacilities of November 18 - 22, 2002 

### RCC Ltd 4452 ltingen Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for Therapeutic Products and the Swiss Agency forthe Environment, Forests and Landscape with respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities -Toxicology

- Environmental Chemistry and

Pharmanalytics Areas ofexpertise* TOX, ACC, MUT, OTH (Safety Pharmacology) ACC, ECT, ENF, EMN, PCT, RES, OTH (Animal metabolism)

The inspections were performed in agreement with the OECD Guidelines for National GLP Inspections and Audits. Itwas found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time they were inspected.

Bern, March 2003 Federal Office of Public Health The Director

Prof. Th. Zeltner

• TOX =Toxicology;ACC =Analyticaland Clinical Chemistry; ECT = Environmental toxicityon aquaticand terrestrial organisms ; ENF =BehaviourInwater, soliand air. Bioaccumulation ; EMN =Studieson effects onmesocosmsand natural ecosystems; MUT "'Mutagenicity; PCT =Physical-chemicaltesting; RES"' Residue studies ; OTH=Other, tobespecified. Page2RCC Study Number848630 DEA/Acid anhydride reaction product
GOOD LABORATORY PRACTICE REPORT



# STATEMENT OF COMPLIANCE

RCC Study Number: Test Item: Study Director: Study Title: 848630 DENAcid anhydride reaction product Mr. Roger Bi:itscher Acute toxicity of DENAcid anhydride reaction product to zebra fish (Brachydanio rerio) 

### in a 96-hour semi-statictest Page3



### This study (with the exception of the pre-experiments as mentioned in the report and the analytical standards as mentioned in the analytical phase report) has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 261 h, 

### 1997 by decision ofthe OECD Council [C(97) 186/Final]. There were no circumstances that may have affected the quality or integrity ofthe data. Study Director: Mr. Roger Bi:itscher

Date:RCC Study Number 848630 DENAcid anhydride reaction product 

# SIGNATURES

Study Director:

Management: REPORT Page4

Mr. Roger

**B~itscher**

Date:

Dr. Uwe MorgenrothRCC Study Number 848630 DEA/Acid anhydride reaction product 

# QUALITYASSURANCE REPORT

RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 ltingen 

### I Switzerland STATEMENT Study Number: Test Item: Study Director: Study Title: 848630 DENAcid anhydride reaction product Mr. Roger Batscher Acute toxicity of DENAcid anhydride reaction product to zebra fish (Brachydanio rerio) in a 96-hoursemi-statictest Page 5

The general facilities and activities are inspected periodically and the results are reported to the responsible person and the management. Study procedures (with the exception of the pre-experiments as mentioned in the report) were periodically inspected. The study plan and this report were audited by the Quality Assurance. The dates are given below.
Dates and Types ofQA Inspections Dates of Reports tothe Study Director and to the Management December 12, 2003 Study plan December 12, 2003 May 05,2004 Process based May 05,2004 (application, test system) October 8/10/11 , 2004 Report October 11, 2004 This statement also confirms that this final report reflects the raw data.
QualityAssurance: 

# f' Ms. Andrea GOdel



# ~~.. 

# .



# ......:·....

**0..~**

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

# N

**~9** 1 

# 2GO

# 4RCC Study Number848630 DEA/Acid anhydride reaction product 

# CONTENTS REPORT Page6



### GLP CERTIFICATE.........................................................................................................2 STATEMENT OF COMPLIANCE ....................................................................................3 SIGNATURES .................................................................................................................4 QUALITY ASSURANCE·..................................................................................................5 CONTENTS ....................................................................................................................6 CONTENTS CONTINUED (TABLES)..............................................................................7 PREFACE .......................................................................................................................8 DATA REQUIREMENTS 

### ITEST GUIDELINES...............................................................9 SUMMARY OF STUDY PLAN AMENDMENTS ...............................................................9 SUMMARY ....................................................................................................................10 1 PURPOSE............................................................................................................11 2 MATERIALS AND METHODS..............................................................................11 2.1 DEFINITIONS .............................................................................................11 2.2 TEST ITEM .................................................................................................12 2.3 ANALYTICAL STANDARD..........................................................................12 2.4 TEST SYSTEM ...........................................................................................12 2.5 STUDY DESIGN .........................................................................................13 2.5.1 Experimental Conditions ................................................................13 2.5.2 Preparation ofTest Solutions and Concentrations
.........................14 2.6 EVALUATIONS ...........................................................................................15 2.6.1 Determination ofthe LC50, LCO, LC100, NOEC and LOEC.............................................................................................15 2.6.2 Water Quality Criteria ....................................................................15 2.6.3 Analysis ofthe Test Item Concentrations....................................... 15 3 RESULTS AND DISCUSSION .............................................................................16 4 TABLES ...............................................................................................................17 ATTACHMENT ..............................................................................................................19 ANALYTICAL PHASE REPORT (Pages 1to 33)RCC Study Number848630 DEA/Acid anhydride reaction product REPORT 

# CONTENTS CONTINUED (TABLES) Page 7



### Table 1: Mortality and visible abnormalities observed in the test fish
........................17 Table 2: Appearance ofthe freshly prepared and old test medium
...........................17 Table 3: pH-values ofthe freshly prepared and old test medium and in the control.........................................................................................18 Table 4: Oxygen concentrations (mg/L) ofthe freshly prepared and old test medium and in the control.
.......................................................18 Table 5: Temperatures 

### CC) ofthe freshly prepared and old test medium and in the control.........................................................................................18RCC Study Number848630 DENAcid anhydride reaction product 

# PREFACE GENERAL Study Title:
Sponsor:
Study Monitor:

Test Facility:

Analytical Phase No.: 

## RESPONSIBILITIES Study Director: Deputy Study Director: Principal Investigatorfor Analytical Phase: Technical Coordinator: Head of RCC QualityAssurance: 

## SCHEDULE Experimental Starting Date: Experimental Completion Date: Study Completion Date: 

## ARCHIVING REPORT

Acute toxicity of DEA/Acid anhydride reaction product to zebra fish (Brachydanio rerio) in a 96-hour semi-static test Rockwoollnternational A/S Hovedgaden 584 DK-2640 Hedehusene Denmark Mrs. Anne Jensen (until end of May 2004) Page 8

Mrs. Lea Tobiassen, DVM (from June 01, 2004) Danish Toxicology Centre KogleAIIe 2 DK-2970 H0RSHOLM Denmark RCC Ltd Environmental Chemistry & Pharmanalytics Zelgliweg 1 CH-4452 ltingen 

### I Switzerland 848631

Mr. Roger Batscher Dr. Armin Peither Dr. Tobias Schoop Mr. Frederic Levrechon Mrs. Iris Wuthrich

May 31, 2004 June 21, 2004 November 19, 2004

RCC Ltd, CH-4452 ltingen, Switzerland will retain the study plan, raw data, a sample of the test item and the final report of the present study for at least ten years. No data will be discarded without the Sponsor's consent.RCC Study Number 848630 DENAcid anhydride reaction product REPORT Page 9 

# DATA REQUIREMENTS I

# TEST GUIDELINES

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline forTesting ofChemicals, No. 203, Fish, Acute Toxicity Test, 1992 EU Commission Directive 92/69/EEC, Part C.1: Acute Toxicity for Fish, 1992



# SUMMARY OF STUDY PLAN AMENDMENTS

First Amendment to Study Plan Concerning: Alteration: Reason: Preface 

### I General Change ofthe Study Monitor Update bythe sponsor. during the study. 2.2 Test Item The batch no. 200501.UN2810 Update ofthe study plan since ofthe test item was additionally the batch no. 200501.UN2810 used for pre-experiments and was additionally used in the analytical purposes. Update of study (for pre-experiments and the expiration date (re-analysis analytical purposes). The test date). item was re-analyzed bythe sponsor. 2.6.3 Analyses ofthe The analysis ofacidic The concentrations ofthe Test Item Concentrations anhydrides, trimellitic and anhydrides in the test item were tetrahydrophthalic anhydrides belowthe limit ofquantification (method no. 4 according to the ofthe analytical method. study plan) was not performed. Therefore, a quantification of the concentrations ofthe anhydrides in the diluted aqueous test medium was not possible.RCC Study Number848630 DEA/Acid anhydride reaction product 

# 1 PURPOSE REPORT Page 11



### The purpose of this 96-hour toxicity test was to evaluate the acute toxicity of the test item DENAcid anhydride reaction product to fish. Zebra fish (Brachydanio rerio) 

### were exposed in a semi-static test to an aqueous test medium containing the test item at a nominal concentration of 100 mg/L under defined conditions (limit test). The limit test was based on the results of a range-finding test and was performed in compliance with the test guidelines to demonstrate that the test item has no toxic effect on fish up to at least this concentration. The test fish were observed for mortality and visible abnormalities. The test method and the test species are recommended by the test guidelines of OECD and EU.



# 2 MATERIALS AND METHODS
2.1 DEFINITIONS LCO: The highest test concentration without a significant number ofdead test organisms. LCSO: The median lethal concentration, i.e. the calculated concentration oftest item which causes death in 50% of the test organisms. LC100: The lowest test concentration at which all test organisms are dead. NOEC (No Observed ,!;ffect Concentration):
LOEC (t:owest Observed ,!;_ffect Concentration): The highest test concentration at which no significant toxic effect on the test organisms is observed.
The lowest test concentration at which a significant toxic effect on the test organisms is observed.RCC Study Number848630 DEA/Acid anhydride reaction product 

## 2.5 STUDY DESIGN
2.5.1 Experimental Conditions REPORT Page 13

One glass aquarium with 5 liters test medium was used for each treatment (the single test concentration and the control). The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification. At the start of the test, 7 fish were introduced into each aquarium in a random order. The loading rate was 0.38 g fish wet weight per liter test medium. The test medium and the control were slightly aerated during the test period. The fish were notfed during the test. A semi-static test with daily test medium renewal was performed to keep the test item concentration in the test medium as constant as possible during the test period of 96 hours. During this semi-static test, the test fish were transferred daily into a clean test vessel with freshly prepared test medium ofthe single concentration. Test duration: 96 hours Watertemperature: 21 - 22

# oc during the test period (Table 5) Light conditions: A 16-hour light to 8-hour dark photoperiod, with a 30-minute ' transition period (light intensity during the light period was approximatelywithin the range of 50 to 500 Lux). Test water: Reconstituted water: analytical grade salts were dissolved in deionized waterto obtain the following nominal concentrations: CaCI2 

### x 2H2 0 2.0 mmoi/L (= 294 mg/L) MgS04 x 7H20 0.5 mmoi/L (= 123 mg/L) NaHC03 0.75 mmoi/L (= 65 mg/L) KCI 0.075 mmoi/L (= 5.8 mg/L) Water Hardness Alkalinity Ratio of Ca : Mg Na:K 

# = = 2.5 mmoi/L (= 250 mg/L as CaC03) 0.8 mmoi/L 4 : 1 (based on molarity) 10 : 1 (based on molarity) The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached.RCC Study Number848630 DENAcid anhydride reaction product REPORT


### 2.5.2 Preparation ofTest Solutions and Concentrations Page 14



### A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test fish up to and including the nominal test item concentration of 100 mg/L. Thus, a single test item concentration of nominal 100 mg/L was tested. Additionally, a control was tested in parallel (test water without addition of the test item). The test medium was prepared before the start of the test and prior to each test medium renewal by mixing 501 - 505 mg of the test item into 5000 ml test water using ultrasonic treatment for 15 minutes and intense stirring for 25 minutes at room temperature. The test medium was freshly prepared just before introduction ofthe fish (= 

### start ofthe test) and just priorto the test medium renewals. The actualconcentration ofthe test item (i.e. ofthe different components ofthe test item) in the test medium was analytically determined (see Section 2.6.3). The limit test was based on the results of a range-finding test and on the results of a preexperiment to determine the solubility of the test item (without GLP). However, concentrations above nominal 100 mg/Lwere not tested according to the guidelines.RCC Study Number848630 DEA/Acid anhydride reaction product 

# 4 TABLES REPORT

Table 1: Mortality and visible abnormalities observed in the test fish Abbreviations ofvisible abnormalities forwhichfish were observed duringthe study: AK: AP: Strongly extended gills Apathy OB: SA: Fish mainly atthe water surface Mucus secretion Page 17

BA: BO: Distended abdomen Fish mainly atthe bottom ofthe aquarium Exophthalmus Convulsions SR: SV: Fish lying on side or back on the bottom Strong ventilation TS: Tumbling during swimming GA: VF: Changed body color KR: Number offish tested at the single concentration and the control: 7 Nominal Number ofaffected fish* I number ofdead fish test item visible abnormalities concentration Observation time (mg/L) 2% hours 24 hours 48 hours 72 hours 96 hours Control 

### 010 010 010 010 0/0

100 

### 010 010 010 010 0/0 LCSO > 100 mg/L * number ofdead fish plus number offish with visible abnormalities

Table 2: Appearance ofthe freshly prepared and old test medium Abbreviations: 0: no remarkable observations, cleartest medium 1: homogeneous dispersion in the water, turbidity observable 2: noticeableturbidity caused bythe test item 3: noticeable coloration caused bythe test item 4: inhomogeneous dispersion ofthe test item 5: precipitation ofthe test item 6: test item at the surface 7: test item lying atthe bottom ofthe aquarium Nominal test item Exposure time concentration Oh 24 h 48 h 72 h 96 h (mg/L) new old new old new old new old

100 0 0 0 0 0 0 0 0RCC Study Number848630 DEA/Acid anhydride reaction product REPORT Page 18

Table 3: pH-values ofthe freshly

**pr~pared** and old test medium and in the control

Nominal test item Exposure time concentration 

### Oh 24 h 48 h 72 h 96 h (mg/L) new old new old new old new old Control 

### 7.7 7.8 7.8 7.9 7.8 7.8 7.8 7.8

100 7.6 7.7 7.5 7.6 7.6 7.6 7.5 7.6

Table 4: Oxygen concentrations (mg/L) ofthe freshly prepared and old test medium and in the control

Nominal test item Exposure time concentration 

### Oh 24 h 48 h 72 h 96 h (mg/L) new old new old new old new old Control 

### 8.6 8.4 8.5 8.3 8.5 8.4 8.6 8.5

100 8.7 8.4 8.4 8.7 8.5 8.5 8.3 8.6

Table 5: Temperatures CC) ofthe freshly prepared and old test medium and in the control

Nominal test item Exposure time concentration 

### Oh 24 h 48 h 72 h 96 h (mg/L) new old new old new old new old Control 

### 22 22 22 22 22 21 22 21

100 22 22 22 22 22 21 22 21RCC Study Number848630 DEA/Acid anhydride reaction product REPORT



# ATTACHMENT

ANALYTICAL PHASE REPORT Page 19

# ANALYTICAL PHASE REPORT Analytical Phase to: ACUTE TOXICITY OF DEAIACID ANHYDRIDE REACTION PRODUCT TO ZEBRA FISH 

### (BRACHYDAN/0 RER/0) IN A 96-HOUR SEMI-STATIC TEST Study Director: Mr. Roger Batscher Principal InvestigatorAnalytical Phase: Dr. Tobias Schoop Completion Date oftheAnalytical Phase Report: November 19, 2004
Test Facility: RCC ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen/Switzerland Identification: RCC Study Number: 848630 RCC Phase Number: 848631

Page 1 to 33RCC Study Number848630 ANALYTICAL PHASE REPORT DEA/Acid anhydride reaction product RCC Phase Number 848631 GOOD LABORATORY PRACTICE 

# STATEMENT OF COMPLIANCE

RCC Study No.: Study Director:

Analytical Phase Number: Test Item: Principal Investigatorfor Analytical Phase: Analytical Phase to: 848630 Mr. Roger

**B~:ltscher** RCC Ltd Environmental Chemistry 

### & Pharmanalytics 848631 DENAcid anhydride reaction product
Dr. Tobias Schoop Acute toxicity of DENAcid anhydride reaction product to zebra fish (Brachydanio rerio) in a 96-hour semi-statictest Page2

The analytical phase (with the exception of the pre-experiments and the standards used for identification ofthe interesting signals as mentioned in the phase report) has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles ofGood Laboratory Practice, as revised in 1997 and adopted November 261 h, 1997 by decision ofthe OECD Council [C(97)186/Final]. Principal Investigatorfor Analytical Phase: Dr. Tobias Schoop



# ar0~tP ......O
..:

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**~v** ......

# .T}
.................... Date: 

### A/
. I 

## Ae;. '"1Cf4t'G 

### !VCJ

**VC~-.-** b 

### :;..y -.1,. L' fRCC Study Number 848630 ANALYTICAL PHASE REPORT Page3 DEA/Acid anhydride reaction product RCC Phase Number848631 

# CONTENTS STATEMENT OF COMPLIANCE ....................................................................................2 CONTENTS ....................................................................................................................3 PREFACE .......................................................................................................................5 GENERAL.......................................................................................................................5 RESPONSIBILITIES........................................................................................................5 SCHEDULE OF ANALYTICAL PHASE ...........................................................................5 1 PURPOSE..............................................................................................................6 2 MATERIALS AND METHODS................................................................................6 2.1 TEST ITEM ............................................................................................................6 2.2 ANALYTICAL PROCEDURE..................................................................................7 2.2.1 Storage .......................................................................................................7 2.2.2 Reagents and Solvents ...............................................................................7 2.2.3 Standard Solutions used for Sample Quantification ....................................7 2.2.4 Preparation ofSpiked TestWater Samples ................................................7 2.2.5 Analysis ofSamples....................................................................................8 2.3 MEASUREMENT CONDITIONS ............................................................................8 2.3.1 Method 1 (diethanolamine) .........................................................................8 2.3.2 Method 2 (dimers, trimers and polymers)....................................................9 2.3.3 Method 3 (tetrahydrophthalic acid and trimellitic acid).................................9 2.4 EVALUATION OF RESULTS ...............................................................................10 2.4.1 DEA/Acid anhydride reaction product on the base ofdiethanolamine
....... 10 2.4.2 DEA/Acid anhydride reaction product on the base ofdimers, trimers and polymers ............................................................................................11 2.4.3 DEA/Acid anhydride reaction product on the base of tetrahydrophthalic acid ..............................................................................12 2.4.4 DEA/Acid anhydride reaction product on the base oftrimellitic acid
.......... 13 3 RESULTS AND DISCUSSION .............................................................................14 3.1 METHOD 1 (DIETHANOLAMINE) ........................................................................14 3.2 METHOD 2 (DIMERS/TRIMERS AND POLYMERS)............................................14 3.3 METHOD 3 (TETRAHYDROPHTHALIC ACID AND TRIMELLITIC ACID)
............ 15 4 TABLES ...............................................................................................................17 Table 1: Example ofcalibration data oftest item standards fordiethanolamine
........ 17 Table 2: Calibration data of test item standards for dimers, trimers and polymers .....................................................................................................18 Table 3: Example of calibration data of test item standards for tetrahydrophthalic acid (cis-4-cyclohexene-1 ,2-dicarbonic acid)
.................. 19 Table 4: Example of calibration data of test item standards for trimellitic acid (1 ,2,4-Benzenetricarboxylic acid) ................................................................20 Table 5: Results obtained for the concentrations of the test item in the test medium on the base ofdiethanolamine.......................................................21RCC Study Number848630 ANALYTICAL PHASE REPORT DEA/Acid anhydride reaction product RCC Phase Number 848631 Table 6: Results obtained for the concentrations of the test item in the test medium on the base ofdilners, trimers and polymers .................................22 Table 7: Results obtained for the concentrations of the test item in the test medium on the base oftetrahydrophthalic acid (cis-4-cyclohexene-1 ,2- dicarbonic acid) ...........................................................................................23 Table 8: Results obtained for the concentrations of the test item in the test medium on the base of trimellitic acid (1 ,2,4-Benzenetricarboxylic acid)............................................................................................................24 5 FIGURES .............................................................................................................25 5.1 Method 1 (diethanolamine) .......................................................................25 Figure 1: HPLC-chromatogram ofstandard solution (low-level).....................................25 Figure 2: HPLC-chromatogram ofstandard solution (high-level) ...................................25 Figure 3: HPLC-chromatogram of biological control sample..........................................26 Figure 4: HPLC-chromatogram ofspiked test water sample..........................................26Page4

Figure 5: HPLC-chromatogram of non-aged treatment sample .....................................27 Figure 6: HPLC-chromatogram ofaged treatment sample ............................................27 5.2 Method 2 (Dimers/trimers and Polymers)..................................................28 Figure 7: HPLC-chromatogram ofstandard solution (low-level).....................................28 Figure 8: HPLC-chromatogram ofstandard solution (high-level) ...................................28 Figure 9: HPLC-chromatogram of biological control sample..........................................29 Figure 10: HPLC-chromatogram ofspiked test water sample........................................29 Figure 11: HPLC-chromatogram ofnon-aged treatment sample ...................................30 Figure 12: HPLC-chromatogram ofaged treatment sample ..........................................30 5.3 Method 3 (tetrahydrophthalic acid and trimellitic acid)...............................31 Figure 13: HPLC-chromatogram ofstandard solution (low/mid-level) ............................31 Figure 14: HPLC-chromatogram ofstandard solution (high-level) .................................31 Figure 15: HPLC-chromatogram of biological control sample........................................32 Figure 16: HPLC-chromatogram ofspiked test water sample........................................32 Figure 17: HPLC-chromatogram of non-aged treatment sample ...................................33 Figure 18: HPLC-chromatogram ofaged treatment sample ..........................................33RCC Study Number 848630 ANALYTICAL PHASE REPORT DEA/Acid anhydride reaction product RCC Phase Number 848631 

# PREFACE
GENERAL Analytical Phase to:

Sponsor:

Study Monitor:

Test Facility:



## RESPONSIBILITIES Study Director: Principal Investigator for analytical phase: Technical Coordinator: Reporting: Head of RCC QualityAssurance: Acute toxicity of DENAcid anhydride reaction product to zebra fish (Brachydanio rerio) in a 96-hoursemi-statictest Rockwoollnternational

### NS Hovedgaden 584 DK-2640 Hedehusene Denmark Mrs. Anne Jensen (until end of May 2004) Mrs. Lea Tobiassen, DVM (from June 01, 2004) Danish Toxicology Centre Kogle Aile 2 DK-2970 H0RSHOLM Denmark RCC Ltd Environmental Chemistry & Pharmanalytics Zelgliweg 1 CH-4452 ltingen/Switzerland

Mr. Roger Batscher
Dr. Tobias Schoop Mrs. Mandy Broda Dr. Tobias Schoop
Mrs. Iris Wuthrich 

## SCHEDULE OF ANALYTICAL PHASE Experimental starting date: Experimental completion date: Completion date ofthe Analytical Phase Report: June 03, 2004 June 21, 2004 November 19, 2004 Page 5RCC Study Number 848630 ANALYTICAL PHASE REPORT Page 7 DEA/Acid anhydride reaction product RCC Phase Number 848631 

## 2.2 ANALYTICAL PROCEDURE 2.2.1 Storage Analysis ofdiethanolamine (method 1) The samples were analysed directly afterthe sampling procedure. Analysis ofdimers, trimers and polymers (method 2) The samples were stored deep-frozen and protected from light until analysis was performed. The stability ofthe frozen samples was tested during non-GLP pre-experiments. Analysis oftetrahydrophthalic acid and trimellitic acid (method 3) The samples were analysed directly afterthe sampling procedure. 

### 2.2.2 Reagents and Solvents Acetonitrile (purity> 99.8 %) Methanol (purity> 99.8 

### %) Nitric acid (purity 65%) Ammoniumdihydrogenphosphate

(purity> 99 %) Purified waterfor HPLC Testwater J.T.Baker, no. 9017 J.T.Baker, no. 8402 J.T.Baker, no. 6080 Fluka, no. 09717 in-house prepared by a water purification system (Millipore) asdescribed in the biological part ofthis study 

### 2.2.3 Standard Solutions used for Sample Quantification Typical example: The test item (109.46 mg) was dissolved in methanol and made up to the mark in a 50 ml volumetric flask to prepare a stock solution of 2189.2 mg/L. Defined volumes of this stock solution were diluted with methanol/water 1:9 (method 2 & 3) respectively methanol/test water 1:9 (method 1) to obtain standard solutions ofthe test item in the range of 10.9 to 219 mg/L. The total calibration range covered by these solutions was used forthe calibration ofall active ingredients using the

**res~ective** methods. 

### 2.2.4 Preparation ofSpiked TestWater Samples To demonstrate the validity ofthe method, untreated test waterwas spiked with the test item. Typical example: The test item (507.2 mg) was dissolved in methanol and made up to the mark in a 50 ml volumetric flask to prepare a stock solution of 10144 mg/L. 0.1 ml ofthis stock solution were diluted with test water to obtain spiked samples with a concentration of 101.44 mg/L. These solutions were freshly prepared and subjected to the same treatment as a sample without any storage.RCC Study Number 848630 ANALYTICAL PHASE REPORT PageS DEA/Acid anhydride reaction product RCC Phase Number 848631 In addition, testwaterwithout the test item was analyzed (analytical blank). 

### 2.2.5 Analysis ofSamples Analysis of diethanolamine (method 1), tetrahydrophthalic acid and trimellitic acid (method 3) The samples were directly analysed without any storage. 900 IJL aliquots ofthe samples were diluted with 100 IJL methanol priorto analysis (dilution factor 1.111). 

### Analysis ofdimers, trimers and polymers (method 2) Treatment samples and control samples were thawed at room temperature for one hour and shaken manually to obtain homogeneous sample solutions. 900 !JL aliquots of the samples were diluted with 100 !JL methanol priorto analysis (dilution factor 1.111). Aliquots ofthe final solutions were analyzed by the respective methods. The results obtained are presented in Table 5 (diethanolamine), Table 6 (dimers, trimers and polymers), Table 7 (tetrahydrophthalic acid) and Table 8 (trimellitic acid).


## 2.3 MEASUREMENT CONDITIONS
2.3.1 Method 1 (diethanolamine) Auto sampler: Pump: Detector: Column: Eluent Flow rate: Run time: Temperature: Injection volume: Detection: Retention time: Merck-Hitachi L-7200 Merck-Hitachi L-6200 732 IC Detector Metrosep Cation 1-2; 125 x 4 mm; 7 1-1m 2 mM HN03 (w

# = 65%)/1 Lwater (milli-Q) 1 mUminute 15 minutes Room temperature

electrochemical approximately 6 minutesRCC Study Number848630 ANALYTICAL PHASE REPORT DEA/Acid anhydride reaction product RCC Phase Number 848631 

### 2.3.2 Method 2 (dimers,

**trime~~** 

### and polymers) Auto sampler: Pump: Detector: Column oven: Column: Eluent: Flow rate: Run time: Temperature: Injection volume: Merck-Hitachi L-7200 Merck-Hitachi L-7100 Merck-Hitachi DAD L-7400 Merck-Hitachi L-7300 Superdex Peptide 10/300 GL methanol/0.035 M NaCI10/90 (v:v) 1 mUminute 25 minutes 40°C in a thermostated oven 100 IJL Detection wave length: 210 nm Retention time: approximately 11-18 minutes



### 2.3.3 Method 3 (tetrahydrophthalic acid and trimellitic acid)
Auto sampler: Pump: Detector: Column: Pre-Column: Varian 9095 Varian ProStar230 Varian 9050 Phenomenex Luna C8 ; 150 X 4.6 mm; 3 IJm Phenomenex C8 ;4 X 3 mm Page9

Eluent: 0.1 M NH4HzP04/ acetonitrile 9 + 1, adjusted to pH 2.5 with H3P04 Flow rate: 0.8 mUminute Run time: 30 minutes Temperature: Room temperature Injection volume: 100 IJL Detection wave length: 210 nm Retention time: -6.5 minutes (Trimellitic acid) -25 minutes (Tetrahydrophthalic acid)RCC Study Number 848630 ANALYTICAL PHASE REPORT Page 10 DEA/Acid anhydride reaction product RCC Phase Number848631 

## 2.4 EVALUATION OF RESULTS 2.4.1 DEAl Acid anhydride reaction producton the base ofdiethanolamine Injected samples were quantified by peak areas with reference to the respective calibration curve. The latter was obtained by correlation of the peak area of the standard solutions to their corresponding concentration in mg/L. The correlation was performed using a potential function given below (equation 1). The results obtained are presented in Table 1. From the calibration curve the concentration x of the test item in an injected sample was calculated by the following equation: 

### x={f (1)
where y 

### = Peak area ofthe test item in injected sample [counts] x 

### = Concentration ofthe test item in injected sample [mg/L] a, b 

# = constants
The concentration ofthe test item in a sample was calculated by equation 2: 

### c=x·F (2) where c 

# = Concentration ofthe test item in the sample [mg/L] x 

# = Amount ofthe test item in injected sample found by equation 1 [mg/L] F 

# = Factor
The recovery ofthe test item in a sample was calculated by equation 3: 

### R=-c- · 100% Cnom where R 

### = Recovery [%] (3)

c 

# = Concentration ofthe test item in the sample found by equation 2 [mg/L] Cnom 

# = Nominal concentration ofthe test item in the sample [mg/L]RCC Study Number 848630 ANALYTICAL PHASE REPORT Page 11 DEA/Acid anhydride reaction product RCC Phase Number 848631 

### 2.4.2 DEAl Acid anhydride reaction product on the base ofdimers, trimers and polymers Injected samples were quantified by peak areas with reference to the respective calibration curve. The latter was obtained by correlation of the peak area of the standard solutions to their corresponding concentration in mg/L. The correlation was performed using a linear function given below (equation 4). The results obtained are presented in Table 2. From the calibration curve the concentration x of the test item in an injected sample was calculated by the following equation: 

### y-a X=-- 

### b 

## (4)
where y 

### = Peak area ofthe test item in injected sample [counts] x 

# = Concentration ofthe test item in injected sample [mg/L] a 

### = y-axis intercept b 

### = Slope
The concentration ofthe test item in a sample was calculated by equation 2: 

### c=x·F (5) where c 

### = Concentration ofthe test item in the sample [mg/L] x 

# = Amount ofthe test item in injected sample found by equation 4 [mg/L] F 

# = Factor
The recovery ofthe test item in a sample was calculated by equation 6: 

### R=-c- · 100% Cnom where R 

### = Recovery[%] (6)

c 

# = Concentration ofthe test item in the sample found by equation 5 [mg/L] Cnom 

# = Nominal concentration ofthe test item in the sample [mg/L]