# RCC Study Number848621

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 1of 17RCC 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 QUALITYASSURANCE 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.....................................................................................................................10 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 STATISTICALANALYSIS .............................................................................................13 8 DATACOMPILATION
....................................-:..............................................................13 9 RESULTS......................................................

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### ..............................................................14 9.1 MORTALITY.........................................................................................................14 9.2 CLINICAL SIGNS.................................................................................................14 9.3 BODYWEIGHTS ..................................................................................................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................................................................................................17RCC 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 Deputyfor Study Director Technical Coordinator Head of RCC Quality Assurance 

## 1.3 SCHEDULE G. Arcelin M.Ott N. Hostettler 

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Experimental Starting Date 13-MA Y-2003 Experimental Completion Date 05-JUN-2003 DeliveryofAnimals 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 StudyCompletion Date 20-MA Y-2003 (females, 2000 mglkg) 22-MA Y-2003 (females, 2000 mglkg) 13-MAY-2003to 03-JUN-2003 (females, 2000 mglkg) 15-MAY-2003to 05-JUN-2003 (females, 2000 mglkg) 15-SEP-2003RCC 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 

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**~~** Dr. H. FankhauserRCC STUDY NUMBER 848621 DEA/Acid anhydride reaction product Report


## 1.6 QUALITY ASSURANCE UNIT
RCC Ltd, Toxicology, CH-4452 ltingen 

### ISwitzerland
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 ofthe 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 thatthis final report reflects the raw data. QualityAssurance: Dr. Z.

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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 February2nd, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November26th, 1997 by decision ofthe OECD Council [C(97)186/Final]. Study Director: 

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## 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 December2001. 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 ofthe 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 athird animal lost body weight (0.6 

### %) between test day 8 and the end of the observation period. The bodyweight of the other animals was within the range commonly recorded forthis 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 14days is: LDso (female rat): greaterthan 2000 mg/kg bodyweightRCC 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 bythe international guidelines as a recommended test system. Source RCC Ltd, LaboratoryAnimal Services CH-4414 FOIIinsdorf 

### I Switzerland Number of animals per group 3females Total number of animals 6females Age when treated 11
- 12 weeks Identification Unique cage number and corresponding color-coded spots on the tail. The animals were marked immediately priorto treatment. Randomization Selected by hand attime of delivery. No computer generated randomization program. Acclimatization Under laboratoryconditions, after health examination. Only animals without anyvisible signs of illness were used forthe 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 ofvehicle 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), awayfrom direct sunlight. Routine hygienic procedures were used to ensure the health and safetyof the personnel. PEG 300 was found to be a suitablevehicle. 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 ofthetest item as supplied bythe 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 approximately3 hours afterdosing. The application volume was 10 mUkg bodyweight. 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 Bodyweights Clinical signs Daily during acclimatization and twice dailyduring days 1-15. On test days 1 (priorto 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 bodyweight (equivalentto at least 324 mg sodium pentobarbitone/kg body weight) and discarded after macroscopic examinations were performed. No organs ortissues were retained. 

## 7 STATISTICAL ANALYSIS No statistical analysis was used. 

## 8 DATA COMPILATION Bodyweights 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. Macroscopicfindings were compiled intothe 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 ofthe 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 bodyweight (0.6 

### o/o) between test day8 and the end ofthe observation period. The bodyweight of the other animals was within the range commonly recorded forthis 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): greaterthan 2000 mg/kg bodyweightRCC 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 aftertreatment. 11 

## .y .y .y .y .y .y

No clinical signs were evident in anyanimal 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 Bodyweights 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 .yRCC 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 macroscopicfindings 2 F 

# s No macroscopicfindings 3 F 

# s No macroscopicfindings 2000 4 F 

# s No macroscopicfindings 5 F 

# s No macroscopicfindings 6 F 

# s No macroscopicfindings S: Scheduled necropsy Page 16RCC STUDY NUMBER 848621 DEA/Acid anhydride reaction product Report


## 11 GLP- CERTIFICATION

The Swiss GLP Monitoring Authorities
Swiss Federal Officeof PublicHealth SwissAgencyforthe Environment, Forests and Landscape **~·** Swissmedlc ' • SwissAgencyfer SWlSSTnedlC Therapeutic Products 

# Statement of GLP Compliance

It is herebyconfirmed that during the period of thefollowingTest Facilitiesof 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 Agencyforthe 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 testlacilitieswere operating in compliance with the Swiss Ordinance relating to Good Laboratory,Practice [RS 813.016.5] at the timetheywere inspected.

Bern, March 2003 Federal Office ofPublic Health The Director

Prof. Th. Zeltner

•TOX =Toxicology:ACC =Analyticaland Clinical Chemistry: ECT=Environmental toxicityon aquaticand terrestrial organisms : ENF =BehaviourInwater, soilandair. Bioaccumulalion : EMN =Studieson effects on mesocosms and natural ecosystems; MUT

### = Mutagenicity; PCT =Physical-chemicaltesting :RES =Residue studies :OTH =

**O~r.** tobespecified. Page 17