# REPORT
StudyTitle ACUTE TOXICITY OF DEAIACID ANHYDRIDE REACTION PRODUCT TO 

### DAPHNIA MAGNA IN A 48-HOUR IMMOBILIZATION TEST Data Requirements ITestGuidelines: OECD No. 202, Part I EU Commission Directive 92/69/EEC, C.2 Study Director: Roger

**B~Uscher** StudyCompletion Date: November 19, 2004 Test Facility: RCC Ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen I Switzerland Sponsor: Rockwool InternationalA/S Hovedgaden 584 DK-2640 Hedehusene Denmark Identification: RCC Study Number: Page 1 of52 848632RCC Study Number848632 DENAcid anhydride reaction product 

# GLP CERTIFICATE REPORT

The Swiss GLP Monitoring Authorities
Swiss Federal Officeof Public Health SwissAgencyforthe Environment, Forests and Landscape / Swissmedic 

## r . SwissAgencyfor 

### SWlSSrnedtc TherapeuucProducts 

# Statement of GLP Compliance

It is hereby confirmed that during the period of thefollowingTest Facilities 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=Analytical and Clinical Chemislly ; ECT=Environmental toxicityon aquaticand terreslrlal organisms ; ENF

### = BehaviourIn water, soiland air. BioaccumulaUon ; EMN=Studieson effects on mesocosms and natural ecosystems; MUT

### =Mutagenicity; PCT

### = Physical-chemicaltesting ; RES=Residue studies ;OTH=Other, tobespecified. Page2RCC Study Number848632 DENAcid anhydride reaction product GOOD LABORATORY PRACTICE REPORT Page 3



# STATEMENT OF COMPLIANCE

RCC Study Number: Test Item: Study Director: Study Title: 848632 DEA/Acid anhydride reaction product Mr. Roger Batscher Acute toxicity of DEA/Acid anhydride reaction product to Daphnia magna 

### in a 48-hour immobilization test 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 2"ct, 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 Batscher

Date:



### J;o~reiM/aJ.J 

# .A5, 2.oof(-RCC Study Number 848632 DEA/Acid anhydride reaction product 

# SIGNATURES

Study Director:

Management: REPORT Page4

Mr. Roger Batscher

Date: 

# JJo

**lrltM~tJ )~,**  **too~**

Dr. Uwe Morgenroth



# .. fL
...... Date:RCC Study Number848632 DEA/Acid anhydride reaction product REPORT Page 5 

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

### I Switzerland STATEMENT RCC Study Number: Test Item: Study Director: Study Title: 848632 DEA/Acid anhydride reaction product Mr. Roger

**B~itscher** Acute toxicity of DEA/Acid anhydride reaction product to Daphnia magna in a 48-hour immobilization test 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 to the Study Director and to the Management December 12, 2003 Study plan December 12, 2003 July 22, 2004 Process based July 22, 2004 (test system, test conditions) October 16/17/18, 2004 Report October 18, 2004 This statement also confirms that this final report reflects the raw data. Quality Assurance:



# ~rs. Andrea GudelRCC Study Number848632 DENAcid anhydride reaction product 

# CONTENTS REPORT Page6



### GLP CERTIFICATE.........................................................................................................2 STATEMENT OF COMPLIANCE ....................................................................................3 SIGNATURES .................................................................................................................4 QUALITYASSURANCE ..................................................................................................5 CONTENTS ....................................................................................................................6 CONTENTS CONTINUED (TABLES)..............................................................................

### ? 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 Immobility, NOEC, ECO, EC50, and EC100 ....................................................................................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 Number848632 DENAcid anhydride reaction product REPORT 

# CONTENTS CONTINUED (TABLES)

Table 1: Effect of DEN Acid anhydride reaction product on the mobility Page 7



### of Daphnia 

### magna....................................................................................... 17 Table 2: Appearance ofthe test medium during the test period ................................17 Table 3: Dissolved oxygen concentrations, pH values, and temperature in the test medium and the control ..............................................................18RCC Study Number848632 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 Page 8

Acute toxicity of

### DEN Acid anhydride reaction product to Daphnia magna in a 48-hour immobilization test 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 KogleAIIe 2 DK-2970 H0RSHOLM Denmark RCC Ltd Environmental Chemistry & Pharmanalytics Zelgliweg 1 CH-4452 ltingen 

### I Switzerland 848633

Mr. Roger

**B~itscher** Dr. Armin Peither Dr. Tobias Schoop Mr. Frederic Levrechon Mrs. Iris Wuthrich

June 07,2004 June 21, 2004 November 19, 2004

RCC Ltd, CH-4452 ltingen, Switzerland will retain the study plan, raw data, a sample ofthe 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 Number848632 DENAcid anhydride reaction product REPORT Page 11 

# 1 PURPOSE

The purpose of the 48-hour toxicity test was to evaluate the effect of the test item DEA/Acid anhydride reaction product on the mobility of 

### Daphnia magna. Young daphnids were exposed in a 48-hour 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 a range-finding test and performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no acute toxic effect on the daphnids up to at least this concentration. The recorded effect was the immobility of the daphnids. The test method and the test species are recommended by the test guidelines ofOECD and EU.



# 2 MATERIALS AND METHODS 2.1 DEFINITIONS Immobilization:



### ECO: EC50: EC100: NOEC (No Observed ,!;ffect Concentration): those organisms which are not able to swim within 15 seconds after gentle agitation of the test container are considered to be immobile the highest test concentration without a significant number of immobilized test organisms the calculated concentration of test item which results in 50% immobilization the lowest test concentration at which all test organisms are immobile the highesttest concentration at which no significanttoxic effect on the test organisms is observedRCC Study Number848632 DEA/Acid anhydride reaction product 

## 2.5 STUDY DESIGN
2.5.1 Experimental Conditions REPORT Page 13

The test was performed in 100 ml glass beakers filled with 50 ml of test medium. The beakers were covered with glass plates to reduce the loss ofwater and to avoid the entry of dust into the solutions. The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification. At the single test concentration and for the control, 20 daphnids were used divided into four replicates offive daphnids each. The daphnids were randomly distributed to the test vessels at initiation ofthetest. The loading rate was less than one daphnia per5 ml oftest solution. A statictest without test medium renewal was performed. Watertemperature: 20 - 21 

# oc during the test period (Table 3). The test was performed in a temperature-controlled room (room temperature continuously monitored). Light conditions: A 16-hour light to 8-hour dark photoperiod (with a 30 minute transition period). Light intensity during the light period was between approximately 520 and 710 Lux. Test duration: 48 hours Test water: Reconstituted test water: analytical grade salts were dissolved in purified waterto obtain the following nominal concentrations: CaCI2 x 2H20 MgS04 x 7H20 NaHC03 KCI Water Hardness Alkalinity Ratio of Ca : Mg Na:K 2.0 mmoi/L (= 294mg/L) 0.5 mmoi/L (= 123mg/L) 0.75 mmoi/L (= 65 mg/L) 0.075 mmoi/L (= 5.8 mg/L) 2.5 mmoi/L (= 250 mg/L as CaC03) 0.8 mmoi/L


### = 4 : 1 (based on molarity) 

# = 1 0 : 1 (based on molarity) The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated. Feeding: None Positive Control: For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control at least once a year. The latest result of the positive control test in 2004 (48-hour EC50: 0.83 mg/L, RCC Study no. 852355) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2004: 48-hour EC50: 0.55 - 1.1 mg/L).RCC Study Number848632 DENAcid anhydride reaction product REPORT


### 2.5.2 Preparation ofTest Solutions and Concentrations

. " Page 14



### A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Daphnia magna 

### 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 waterwithout addition ofthe test item). The test medium was prepared immediately before the start ofthe test by mixing 50.2 mg of the test item into 500 ml test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The actual concentration 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/L were not tested according to the EU Commission Directive 92/69/EEC.RCC Study Number848632 DENAcid anhydride reaction product 

# 4 TABLES REPORT Page 17



### Table 1: Effect of DEN Acid anhydride reaction product on the mobility of Daphnia magna
Nominal No. of Immobilized test item daphnids daphnids after concentration tested 24 hours (mg/L) No. % Control 20 0 0

100 20 0 0



### Table 2: Appearance ofthe test medium during the test period
Abbreviations: 0: no remarkable observations, cleartest medium 1: homogeneous dispersion in the water, turbidity observable 2: noticeable turbidity caused by the test item 3: noticeable coloration caused bythe test item 4: inhomogeneous dispersion ofthe test item 5: precipitation ofthetest item 6: test item at the surface 7: test item lying atthe bottom ofthe test beaker Nominal test item Exposure time concentration (mg/L) 0 hours 24 hours

100 0* 0 * few fine particles observed Immobilized daphnids after 48 hours No. %

0 0 0 0

48 hours 0RCC Study Number848632 DEA/Acid anhydride reaction product REPORT



### Table 3: Dissolved oxygen

**concent~.ations,** 

### pH values, and temperature in the test medium and the control

Nominal Page 18

test item Start (0 hours) End (48 hours) concentration pH Oxygen Temperature pH Oxygen Temperature (mg/L) (mg/L) (OC) (mg/L) (OC) Control 7.9 9.2 20 7.9 8.6 21

100 7.4 9.1 20 7.8 8.6 21RCC Study Number848632 DENAcid anhydride reaction product REPORT



# ATTACHMENT

ANALYTICAL PHASE REPORT Page 19

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

### DAPHNIA MAGNA IN A 48-HOUR IMMOBILIZATION TEST Study Director: Mr. Roger

**B~Hscher** Principal InvestigatorAnalytical Phase: Dr. Tobias Schaap Completion Date ofthe Analytical Phase Report: November 19, 2004 Test Facility: RCC ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen/Switzerland Identification: RCC Study Number: 848632 RCC Phase Number: 848633

Page 1 to 33RCC Study Number848632 ANALYTICAL PHASE REPORT Page2 DENAcid anhydride reaction product RCC Phase Number 848633 GOOD LABORATORY PRACTICE 

# STATEMENT OF COMPLIANCE

RCC Study No.: Study Director:

Analytical Phase Number: Test Item: Principal Investigatorfor Analytical Phase: Analytical Phase to: 848632 Mr. Roger Batscher RCC Ltd Environmental Chemistry & Pharmanalytics 848633 DENAcid anhydride reaction product Dr. Tobias Schoop Acute toxicity of DEN Acid anhydride reaction product to Daphnia magna in a 48-hour immobilization test 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 2"d, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles ofGood Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision ofthe OECD Council [C(97)186/Final]. Principal Investigatorfor Analytical Phase: Dr. Tobias Schoop 

# .l.?.-.
..

**~**

# ..................... Date: .tf/OVfL-(.)y 

## 41 r 

## ?_roC,RCC Study Number848632 ANALYTICAL PHASE REPORT Page 3 DEA/Acid anhydride reaction product RCC Phase Number 848633 

# 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 Test Water 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 for diethanolamine
........ 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 Number 848632 ANALYTICAL PHASE REPORT DEA/Acid anhydride reaction product RCC Phase Number848633 Table 6: Results obtained for the concentrations of the test item in the test medium on the base ofdihlers, 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) ...........................................................................................23Page4

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 watersample..........................................26 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 watersample........................................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-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 848632 ANALYTICAL PHASE REPORT Page 5 DEA/Acid anhydride reaction product RCC Phase Number 848633 

# PREFACE GENERAL Analytical Phase to: Acute toxicity of DEA/Acid anhydride reaction product to Daphnia magna in a 48-hour immobilization test Sponsor:

Study Monitor:

Test Facility:



## RESPONSIBILITIES Study Director: Principal Investigator for analytical phase: Technical Coordinator: Reporting: Head of RCC QualityAssurance: Rockwoollnternational A/S 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

**B~itscher** 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 08, 2004 June 21, 2004 November 19, 2004RCC Study Number 848632 ANALYTICAL PHASE REPORT Page 16 DEA/Acid anhydride reaction product RCC Phase Number 848633
Concurrent with the sample analysis, recoveries of spiked test water samples at relevant concentrations (101 mg test item/L) were performed in duplicate. The average concentration was found to be 97% of the spiked values (n 

# =4). No correction for any losses during the analytical procedure was made. The routine limit ofquantification (LOQ) for the test item in test medium was derived from the lowest standard solution, which fits into the calibration curve: the value is 10.9 mg/L. With a factor of 1.111 from sample dilution a LOQ ofapproximately 10 mg/L ofthe test item results. The limit of detection (LOD) was calculated to be at 2 mg/L of the test item (1 fifth of the LOQ). The biological control samples and an analyzed analytical blank (test water) did not affect the HPLC-chromatogram at the retention time ofthe test item. The average concentrations found in the treatment samples were 98 % of the nominal concentrations at test start and 99 % ofthe nominal concentrations attest end. It can be seen from Table 8 that trimellitic acid was stable during the performance of the biological test. Typical HPLC chromatograms are shown in the attached Figure 13 to Figure 18.