RCC Study Number 848621 

DEA/Acid anhydride reaction 

product: 

Acute Oral Toxicity Study in Rats 

Report 

Author: G. Arcelin 

Sponsor: Rockwool International A/S 

Hovedgaden 584 

DK-2640 Hedehusene I Denmark 

Page 1 of 17 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

Table of contents 

Report Page2 

1 PREFACE ....................................................................................................................... 3 

1.1 GENERAL ............................................................................................................... 3 

1.2 RESPONSIBILITIES .............................................................................................. 3 

1.3 SCHEDULE ............................................................................................................ 3 

1.4 ARCHIVING ........................................................................................................... 4 

1.5 SIGNATURE PAGE ............................................................................................... 5 

1.6 QUALITY ASSURANCE UNIT ................................................................................ 6 

1.7 STATEMENT OF COMPLIANCE ........................................................................... 7 

1.8 TEST GUIDELINES ............................................................................................... 8 

1.9 ANIMAL WELFARE ................................................................................................ 8 

2 SUMMARY ...................................................................................................................... 9 

3 CONCLUSION ................................................................................................................ 9 

4 PURPOSE ..................................................................................................................... 1 0 

5 MATERIALS AND METHODS ....................................................................................... 10 

5.1 TEST SYSTEM .................................................................................................... 1 0 

5.2 HUSBANDRY ....................................................................................................... 10 

5.3 TEST ITEM .......................................................................................................... 11 

5.4 VEHICLE .............................................................................................................. 11 

5.5 DOSE FORMULATION ........................................................................................ 12 

5.6 TREATMENT ....................................................................................................... 12 

5.7 OBSERVATIONS ................................................................................................. 12 

6 PATHOLOGY ................................................................................................................ 13 

6.1 NECROPSY ......................................................................................................... 13 

7 STATISTICAL ANALYSIS ............................................................................................. 13 

8 DATACOMPILATION ................................... .-: .............................................................. 13 

9 RESUL TS ...................................................... :~ .............................................................. 14 

9.1 MORTALITY ......................................................................................................... 14 

9.2 CLINICAL SIGNS ................................................................................................. 14 

9.3 BODY WEIGHTS .................................................................................................. 14 

9.4 MACROSCOPIC FINDINGS ................................................................................ 14 

9.5 MEDIAN LETHAL DOSE ...................................................................................... 14 

10 INDIVIDUAL FINDINGS ................................................................................................ 15 

10.1 MORTALITY I CLINICAL SIGNS .......................................................................... 15 

10.2 BODY WEIGHTS ................................................................................................. 15 

10.3 MACROSCOPIC FINDINGS ................................................................................ 16 

11 GLP-CERTIFICATION ................................................................................................ 17 
RCC STUDY NUMBER 848621 

DEAl Acid anhydride reaction product 

Report 

1 PREFACE 

1.1 GENERAL 

Title DEAl Acid anhydride reaction product: 

Acute Oral Toxicity Study in Rats 

Sponsor Rockwoollnternational A/S 

Hovedgaden 584 

Study Monitor 

Test Facility 

DK-2640 Hedehusene I Denmark 

Mr. Flemming A. Simonsen, DVM 

Danish Toxicology Centre 

2 Kogle Aile 

DK-2970 Hoersholm I Denmark 

RCC Ltd 

Toxicology 

Wolferstrasse 4 

CH-4414 Fullinsdorf I Switzerland 

1.2 RESPONSIBILITIES 

Study Director 

Deputy for Study Director 

Technical Coordinator 

Head of RCC Quality 

Assurance 

1.3 SCHEDULE 

G. Arcelin 

M.Ott 

N. Hostettler 

1. w uth rich 

Experimental Starting Date 13-MA Y -2003 

Experimental Completion Date 05-JUN-2003 

Delivery of Animals 13-MA Y -2003 (females, 2000 mglkg) 

15-MA Y -2003 (females, 2000 mglkg) 

Page3 

Acclimatization 13-MA Y -2003 to 19-MA Y -2003 (females, 2000 mglkg) 

15-MA Y -2003 to 21-MA Y -2003 (females, 2000 mglkg) 

Treatment 

Observation 

Study Completion Date 

20-MA Y -2003 (females, 2000 mglkg) 

22-MA Y -2003 (females, 2000 mglkg) 

13-MAY-2003 to 03-JUN-2003 (females, 2000 mglkg) 

15-MAY-2003 to 05-JUN-2003 (females, 2000 mglkg) 

15-SEP-2003 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

1.4 ARCHIVING 

Report Page4 

RCC Ltd (CH-4452 ltingen I Switzerland) will retain the study plan, raw data, a sample of test 

item(s) and the final report of the present study for at least ten years. No ·data will be 

discarded without the Sponsor's written consent. 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

1.5 SIGNATURE PAGE 

Study Director: 

Management: 

Report PageS 

G.A/N 

... b.~Jid£& .. ! . date: /J~oJ 

~~ Dr. H. Fankhauser 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

Report 

1.6 QUALITY ASSURANCE UNIT 

RCC Ltd, Toxicology, CH-4452 ltingen I Switzerland 

STATEMENT 

RCC STUDY NUMBER : 

TEST ITEM 

STUDY DIRECTOR 

TITLE 

848621 

DEAl Acid anhydride reaction product 

G. Arcelin 

DEAl Acid anhydride reaction product: 

Acute Oral Toxicity Study in Rats 

Page6 

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 trial formulation, were periodically inspected. The 

study plan and this report were audited by the Quality Assurance Unit. The dates are given 

below. 

Dates and Types of Dates of Reports to the Study Director 

QAU Inspections and to Management 

08-MA Y -2003 Study Plan 08-MAY-2003 

20-MA Y -2003 Process Based (Test 20-MA Y -2003 

System, Test Item, Dose Preparation, 

Treatment, Raw Data) 

01-JUL-2003 Report 01-JUL -2003 

This statement also confirms that this final report reflects the raw data. 

Quality Assurance: Dr. Z. ~ratoljic-Melkay 

fJ .~Ic fJ,tJ(}f,nJO ............... [~·······~~.:~ date: )![". 09. ·wo3 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

Report 

GOOD LABORATORY PRACTICE 

1.7 STATEMENT OF COMPLIANCE 

RCC STUDY NUMBER : 

TEST ITEM 

STUDY DIRECTOR 

TITLE 

848621 

DEAl Acid anhydride reaction product 

G. Arcelin 

DEAl Acid anhydride reaction product: 

Acute Oral Toxicity Study in Rats 

Page? 

The stability of the test item in PEG 300 is unknown. The formulation trials were performed 

before the study initiation date. Therefore, they are excluded from this statement. 

This study 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 26th, 1997 by decision of the OECD Council [C(97)186/Final]. 

Study Director: G./? 

.lz..I!.L.. 

date: ;(·;;;:·~ ~ 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

1.8 TEST GUIDELINES 

Report PageS 

The study procedures described in this report meet or exceed the requirements of the 

following guidelines: · 

OECD Guidelines for the Testin~ of Chemicals, Number 423 .. Acute Oral Toxicity- ~cute 

Toxic Class Method .. , adopted 17 December 2001. 

Directive 96/54/EEC, 8.1 tris .. Acute Oral Toxicity-Acute Toxic Class Method .. , September 30, 

1996. 

1.9 ANIMAL WELFARE 

This study was performed in an AAALAC-approved laboratory in accordance with the Swiss 

Animal Protection Law under license no. 34. 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

2 SUMMARY 

Report Page 9 

Six female HanBrl: WIST (SPF) rats were treated with DEA/Acid anhydride reaction product 

by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was 

diluted in vehicle (PEG 300) at a concentration of 0.2 g/mL and administered at a volume 

dosage of 10 mUkg. · 

The animals were examined daily during the acclimatization period and mortality, viability 

and clinical signs were recorded. All animals were examined for clinical signs at approximately 1 , 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. 

Mortality/viability was recorded twice daily during test days 1-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied 

and examined macroscopically. 

All animals survived until the end of the study period. 

No clinical signs were observed during the course of the study. 

One animal showed a loss of body weight (2.3 o/o) and another animal did not gain body 

weight during the first observation week. Both animals recovered whereas a third animal lost 

body weight (0.6 %) between test day 8 and the end of the observation period. 

The body weight of the other animals was within the range commonly recorded for this strain 

and age. 

No macroscopic findings were recorded at necropsy. 

3 CONCLUSION 

The median lethal dose of DEAl Acid anhydride reaction product after single oral 

administration to female rats, observed over a period of 14 days is: 

LDso (female rat): greater than 2000 mg/kg body weight 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

4 PURPOSE 

Report Page 10 

The purpose of this study was to assess the acute toxicity of DEA/Acid anhydtide reaction 

product when administered by a single oral gavage to rats, followed by an observation period 

of 14 days. 

This study provides information for both hazard assessment and hazard classification 

purposes. 

5 MATERIALS AND METHODS 

5.1 TEST SYSTEM 

Test system Rat, HanBrl: Wist (SPF) 

Rationale Recognized by the international guidelines as a 

recommended test system. 

Source RCC Ltd, Laboratory Animal Services 

CH-4414 FOIIinsdorf I Switzerland 

Number of animals per group 3 females 

Total number of animals 6 females 

Age when treated 11 - 12 weeks 

Identification Unique cage number and corresponding color-coded spots 

on the tail. The animals were marked immediately prior to 

treatment. 

Randomization Selected by hand at time of delivery. 

No computer generated randomization program. 

Acclimatization Under laboratory conditions, after health examination. Only 

animals without any visible signs of illness were used for the 

study. 

5.2 HUSBANDRY 

Room no. 

Conditions 

Accommodation 

1 04 I RCC Ltd, FOIIinsdorf 

Standard Laboratory Conditions. Air-conditioned with 1 0-15 

air changes per hour, and continuously monitored environment with target ranges for room temperature 22 ± 3 oc and 

for relative humidity between 30-70 % (values above 70 o/o 

during cleaning process possible), 12 hours fluorescent light/ 

12 hours dark, music during the light period. 

In groups of three in Makrolon type-4 cages with wire mesh 

tops and standard softwood bedding flignocel' Schill AG, 

CH-4132 Muttenz/Switzerland). 

RCC STUDY NUMBER 848621 

DEAl Acid anhydride reaction product 

Report Page 12 

Stability of vehicle 

Storage conditions 

Safety precautions 

Stable under storage conditions; 

expiration date: 14-NOV-2003 

In the original container, at room temperature 

(range of 20 ± 3 °C), away from direct sunlight. 

Routine hygienic procedures were used to ensure the health 

and safety of the personnel. 

PEG 300 was found to be a suitable vehicle. 

The vehicle was chosen after a non-GLP solubility trial which was performed before the 

study initiation date. This trial formulation is excluded from the GLP statement of compliance. 

5.5 DOSE FORMULATION 

Dose levels are in terms of the test item as supplied by the sponsor. 

The dose formulations were made shortly before each dosing occasion using a magnetic 

stirrer as homogenizer. 

The test item was weighed into a tared glass beaker on a suitable precision balance and the 

vehicle added (weight:volume). 

Homogeneity of the test item in the vehicle was maintained during administration using a 

magnetic stirrer. 

5.6 TREATMENT 

The animals received a single dose of the test item by oral gavage administration at 

2000 mg/kg body weight after being fasted for approximately 17 hours (access to water was 

permitted). Food was provided again approximately 3 hours after dosing. 

The application volume was 1 0 mUkg body weight. 

Rationale: Oral administration was considered to be an appropriate application method as it 

is a possible route of human exposure during manufacture, handling and use of the test 

item. 

5.7 OBSERVATIONS 

Mortality I Viability 

Body weights 

Clinical signs 

Daily during acclimatization and twice daily during days 1-15. 

On test days 1 (prior to administration), 8 and 15. 

Daily during acclimatization and at approximately 1, 2, 3 and 

5 hours after administration on test day 1. Once daily during 

days 2-15. 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

6 PATHOLOGY 

6.1 NECROPSY 

Report Page 13 

All animals were killed at the end of the observation period by an intraperitoneal injecUon of 

Vetanarcol at a dose of at least 2.0 mUkg body weight (equivalent to at least 324 mg sodium 

pentobarbitone/kg body weight) and discarded after macroscopic examinations were 

performed. No organs or tissues were retained. 

7 STATISTICAL ANALYSIS 

No statistical analysis was used. 

8 DATA COMPILATION 

Body weights were recorded on-line. 

Clinical signs were recorded on data sheets. 

Mortality/viability were compiled into the RCC computer system during recording and/or 

recorded on data sheets. 

Macroscopic findings were compiled into the RCC computer system during recording. 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

9 RESULTS 

9.1 MORTALITY 

No deaths occurred during the study. 

9.2 CLINICAL SIGNS 

Report 

No clinical signs were observed during the course of the study. 

9.3 BODY WEIGHTS 

Page 14 

One animal showed a loss of body weight (2.3 o/o) and another animal did not gain body 

weight during the first observation week. Both animals recovered whereas a third animal lost 

body weight (0.6 o/o) between test day 8 and the end of the observation period. 

The body weight of the other animals was within the range commonly recorded for this strain 

and age. 

9.4 MACROSCOPIC FINDINGS 

No macroscopic findings were recorded at necropsy. 

9.5 MEDIAN LETHAL DOSE 

The median lethal dose of DEA/Acid anhydride reaction product after single oral 

administration to female rats, observed over a period of 14 days is: 

LDso (female rat): greater than 2000 mg/kg body weight 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

Report 

10 INDIVIDUAL FINDINGS 

10.1 MORTALITY I CLINICAL SIGNS 

Test days 

Dose An. Sex Signs 1 2 3 

mg/kg No. 1* 2* 3* 5* 

2000 1 F No clinical .y .y .y .y .y .y 

signs 

2 F No clinical .y .y .y .y .y .y 

signs 

3 F No clinical .y .y .y .y .y .y 

signs 

2000 4 F No clinical .y .y .y .y .y .y 

signs 

5 F No clinical .y .y .y .y .y .y 

signs 

6 F No clinical .y .y .y .y .y .y 

signs 

Key: .Y noted 

4 5 6 7 8 9 10 

.y .y .y .y .y .y .y 

.y .y .y .y .y .y .y 

.y .y .y .y .y .y .y 

.y .y .y .y .y .y .y 

.y .y .y .y .y .y .y 

.y .y .y .y .y .y .y 

* Examinations were performed approximately 1, 2, 3 and 5 hours after treatment. 

11 

.y 

.y 

.y 

.y 

.y 

.y 

No clinical signs were evident in any animal during the acclimatization period. 

10.2 BODY WEIGHTS 

Dose An. Sex Day 1 DayS Day 15 

m_g/kg No. (treatmentl_ 

2000 1 F 190.6 197.5 200.9 

2 F 183.0 178.7 184.9 

3 F 193.6 203.8 202.6 

2000 4 F 196.4 205.6 218.5 .. 

5 F 183.4 195.2 201.9 

6 F 199.5 200.1 214.1 

Body weights are presented in grams 

Page 15 

12 13 14 15 

.y .y .y .y 

.y .y .y .y 

.y .y .y .y 

.y .y .y .y 

.y .y .y .y 

.y .y .y .y 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

Report 

10.3 MACROSCOPIC FINDINGS 

Dose An. Sex Mode Findings 

mg/kg No. of 

death 

2000 1 F s No macroscopic findings 

2 F s No macroscopic findings 

3 F s No macroscopic findings 

2000 4 F s No macroscopic findings 

5 F s No macroscopic findings 

6 F s No macroscopic findings 

S: Scheduled necropsy 

Page 16 
RCC STUDY NUMBER 848621 

DEA/Acid anhydride reaction product 

Report 

11 GLP- CERTIFICATION 

The Swiss GLP Monitoring Authorities 

Swiss Federal 

Office of 

Public Health 

Swiss Agency for the 

Environment, Forests 

and Landscape 

~· Swissmedlc 

' • Swiss Agency fer SWlSSTnedlC Therapeutic Products 

Statement of GLP Compliance 

It is hereby confirmed that 

during the period of 

the following Test 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 for the 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 of expertise* 

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. It was found that the aforementioned test lacilities 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 Chemistry: ECT =Environmental toxicity on aquatic and terrestrial organisms : ENF = Behaviour In water, soil and air. Bioaccumulalion : EMN =Studies on effects on mesocosms and natural ecosystems; MUT = Mutagenicity; PCT = Physical-chemical testing : RES = Residue studies : OTH = O~r. to be specified. 

Page 17