RCC Study Number 848622 

DEA/Acid anhydride reaction 

product: 

Acute Dermal Toxicity Study in Rats 

Report 

Author: G. Arcelin 

Sponsor: Rockwoollnternational A/S 

Hovedgaden 584 

DK-2640 Hede;husene I Denmark 

Page 1 of 17 
RCC STUDY NUMBER 848622 

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 OF RESULTS ............................................................................................... 9 

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

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

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

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

5.2 HUSBANDRY ....................................................................................................... 1 0 

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

5.4 TEST ITEM PREPARATION ................................................................................ 11 

5.5 TREATMENT ....................................................................................................... 12 

5.6 OBSERVATIONS ................................................................................................. 12 

6 PATHOLOGY ................................................................................................................ 12 

6.1 NECROPSY ......................................................................................................... 12 

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

8 DATACOMPILATION ................................................................................................... 13 

9 RESUL TS ...................................................................................................................... 14 

9.1 MORTALITY ......................................... .-~ .............................................................. 14 

9.2 CLINICAL I LOCAL SIGNS .................. ~~ .............................................................. 14 

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

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

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

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

10.1 CLINICAL SIGNS ................................................................................................. 15 

10.2 BODY WEIGHTS ................................................................................................. 16 

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

11 GLP- CERTIFICATION ................................................................................................ 17 
RCC STUDY NUMBER 848622 

DEAl Acid anhydride reaction product 

Report 

1 PREFACE 

1.1 GENERAL 

Title DEAl Acid anhydride reaction product: 

Acute Dermal 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 

w olferstrasse 4 

CH-4414 FOIIinsdorf 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. Wuthrich 

Experimental Starting Date 13-MA Y -2003 

Experimental Completion Date 03-JUN-2003 

Delivery of Animals 13-MA Y -2003 

Acclimatization 

Treatment 

Observation 

Study Completion Date 

13-MA Y -2003 to 19-MA Y -2003 

20-MA Y -2003 

13-MAY-2003 to 03-JUN-2003 

17 -SEP-2003 

Page3 
RCC STUDY NUMBER 848622 

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 848622 

DEA/Acid anhydride reaction product 

1.5 SIGNATURE PAGE 

Study Director: 

Management: 

Report PageS 

G.~ 

date: 

/Q ... 

(f~·i-::iJ~ 

~~~ 

~~ Dr. H. Fankhauser 

£~~/~t?J 
RCC STUDY NUMBER 848622 

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 

848622 

DEA/Acid anhydride reaction product 

G. Arcelin 

DEAl Acid anhydride reaction product: 

Acute Dermal 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 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-MA Y -2003 

20-MAY-2003 Test System, Test item, 20-MA Y -2003 

Treatment, Raw Data 

25-JUN-2003 Report 25-J U N-2003 

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

Quality Assurance: 

date: A1-St:P-20o3 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

Report 

GOOD LABORATORY PRACTICE 

1.7 STATEMENT OF COMPLIANCE 

RCC STUDY NUMBER : 

TEST ITEM 

STUDY DIRECTOR 

TITLE 

848622 

DEAl Acid anhydride reaction product 

G. Arcelin 

DEAl Acid anhydride reaction product: 

Acute Dermal Toxicity Study in Rats 

Page? 

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: 
RCC STUDY NUMBER 848622 

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 Testing of Chemicals, Section 4, Number 402 .. Acute Dermal Toxicity .. , 

adopted February 24, 1987. · 

Directive 92/69/EEC, 8.3 ... Acute Toxicity-Dermal .. , July 31, 1992. 

1.9 ANIMAL WELFARE 

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

Animal Protection Law under license no. 32. 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

Report 

2 SUMMARY OF RESULTS 

Page9 

Five male and five female HanBrl: WIST (SPF) rats were treated with DEA/Acid anhydride 

reaction product at 2000 mg/kg by dermal application. The test item was applied undiluted as 

delivered by the Sponsor. The application period was 24 hours. 

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. 

No deaths occurred during the study. 

No clinical signs were observed during the observation period. 

Three female animals showed a loss of body weight {1.5 °/o to 4.7 %) during the first 

observation week and recovered from test day 8 to 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 observed at necropsy. 

3 CONCLUSION 

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

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

LDso (rat): greater than 2000 mg/kg body weight 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

4 PURPOSE 

Report Page 10 

The purpose of this study was to assess the acute dermal toxicity of DEA/AC"id anhydride 

reaction product when administered to rats by a single semi-occlusive dermai application, 

followed by an observation period of 14 days. 

This study should provide a rational basis for risk assessment. 

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 Fullinsdorf I Switzerland 

Number of animals per group 5 males and 5 females 

Total number of animals 5 males and 5 females 

Age when treated Males: 8 - 9 weeks 

Females: 11 - 12 weeks 

Identification By unique cage number and corresponding color-coded 

spots on the tail. The animals were marked immediately prior 

to treatment. 

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 

104 I RCC Ltd, FOJiinsdorf 

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 % 

during cleaning process possible), 12 hours fluorescent light/ 

12 hours dark, music during the light period. 

RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

Report Page 12 

5.5 TREATMENT 

One day before treatment, the backs of the animals were clipped with an electric clipper, 

exposing an area of approximately 1 0 °/o of the total body surface. 

Only those animals without injury or irritation on the skin were used in the test. 

On test day 1 , the test item was applied at a dose of 2000 mg/kg body weight on a patch. 

The patch with the test item was applied on the intact skin and covered with a semi-occlusive 

dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive 

bandage. 

Application amount/kg body weight: 2 g 

Twenty-four hours after the application the dressing was removed and the skin was flushed 

with lukewarm tap water and dried with disposable paper towels. Thereafter, the reaction 

sites were assessed. 

Rationale Dermal administration was used as this is one possible route 

of human exposure during manufacture, handling and use of 

the test item. 

5.6 OBSERVATIONS 

Mortality I Viability 

Body weights 

Clinical signs 

6 PATHOLOGY 

6.1 NECROPSY 

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. 

All animals were killed at the end of the observation period by an intraperitoneal injection 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. 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

Report 

7 STATISTICAL ANALYSIS 

No statistical analysis was used. 

8 DATA COMPILATION 

Body weights were recorded on-line. 

Clinical signs were recorded on data sheets. 

Page 13 

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 848622 

DEA/Acid anhydride reaction product 

9 RESULTS 

9.1 MORTALITY 

No deaths occurred during the study. 

Report 

9.2 CLINICAL I LOCAL SIGNS 

No systemic or local signs of toxicity were observed during the study period. 

9.3 BODY WEIGHTS 

Page 14 

Three female animals showed a loss of body weight (1.5 °/o to 4.7 o/o) during the first 

observation week and recovered from test day 8 to 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 observed at necropsy. 

9.5 MEDIAN LETHAL DOSE 

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

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

LDso (rat): greater than 2000 mg/kg body weight 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

Report 

10 INDIVIDUAL FINDINGS 

10.1 CLINICAL SIGNS 

Sex/ Animal Signs Test Day 

Dose No. 

1 2 

1* 2* 3* 5* 

male/ 1 No clinical signs " " " " " 2000 

" " " " " mg/kg 2 No clinical signs 

3 No clinical signs 

4 No clinical signs " " " " " 

5 No clinical signs " " " " " 

female 6 No clinical signs " " " " " " " " " " /2000 

" " " " " mg/kg 7 No clinical signs 

8 No clinical signs 

9 No clinical signs " " " " " 

10 No clinical signs " " " " " 

Key: " noted " " " " " 

3 4 5 6 7 8 9 

" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 

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

10 

" 

" 

" 

" 

" 

" 

" 

" 

" 

" 

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

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11 12 13 14 15 

" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

10.2 BODY WEIGHTS 

Body weight in grams: 

Sex/ Dose Animal No. 

male I 2000 mg/kg 1 

2 

3 

4 

5 

female I 2000 mg/kg 6 

7 

8 

9 

10 

Report 

Day of Treatment 

246.0 

251.6 

257.3 

247.9 

250.1 

187.0 

191.6 

189.4 

196.4 

190.1 

10.3 MACROSCOPIC FINDINGS 

Sex/ Dose Animal No. Mode of Death 

male I 2000 mg/kg 1 Scheduled necropsy 

2 Scheduled necropsy 

3 Scheduled necropsy 

4 Scheduled necropsy 

5 Scheduled necropsy 

female I 2000 mg/kg 6 Scheduled necropsy 

7 Scheduled necropsy 

8 Scheduled necr9psy 

9 Scheduled necropsy 

10 Scheduled necropsy 

Page 16 

DayS Day 15 

267.7 285.4 

266.0 281.2 

278.8 312.2 

265.3 283.2 

260.9 278.5 

195.9 205.9 

194.4 195.7 

186.3 201.3 

187.1 194.2 

187.2 196.3 

Findings 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 

No macroscopic findings. 
RCC STUDY NUMBER 848622 

DEA/Acid anhydride reaction product 

Report 

11 GLP- CERTIFICATION 

The Swiss GLP Monitoring Authorities 

SWiss Federal 

Office of 

Public Health 

Swiss Agency for the .J·. Swlssmedic 

Environment, Forests r . Swiss Agency for 

andlandscape SWlSSmedlC TherapeulicProducts 

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 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 Chemistry; ECT = Environmental toxicity on aquatic and terrestrial organisms ; ENF = Behaviour in water, soil and alr. Bloaccumutatfon ; EMN :a: Studies on effects on mesocosms and natural ecosystems; MUT = Mutagenicity; PCT = Physical-chemical testing ; RES = Residue studies : OTH = Oth~r. to be specified. 

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