Acute Oral Toxicity Study in Rats 

Report 

Author: G. Arcelin 

Sponsor: Olof Beckmann 

Page 1 of 19 
Report Page2 

TABLE OF CONTENTS 

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 GLP TOXICOLOGY ........................................................ 6 

1. 7 STATEMENT OF COMPLIANCE ........................................................................... 7 

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

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

1.10 SUMMARY OF STUDY PLAN AMENDMENT ........................................................ 8 

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

3 CONCLUSION .............................................................................................................. 10 

4 PURPOSE .................................................................................................................... 11 

5 MATERIALS AND METHODS ...................................................................................... 11 

5.1 TEST SYSTEM .................................................................................................... 11 

5.2 HUSBANDRY ...................................................................................................... 11 

5.3 TEST ITEM .......................................................................................................... 12 

5.4 VEHICLE ............................................................................................................. 12 

5.5 DOSE FORMULATION ........................................................................................ 13 

5.6 TREATMENT ....................................................................................................... 13 

5.7 OBSERVATIONS ................................................................................................ 13 

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

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

7 STATISTICAL ANALYSIS ............................................................................................. 14 

8 DATA COMPILATION .................................................................................................. 14 

9 RESULTS ..................................................................................................................... 15 

9.1 MORTALITY ........................................................................................................ 15 

9.2 CLINICAL SIGNS ................................................................................................ 15 

9.3 BODY WEIGHTS ................................................................................................. 15 

9.4 MACROSCOPIC FINDINGS ................................................................................ 15 

9.5 MEDIAN LETHAL DOSE ..................................................................................... 15 

10 INDIVIDUAL FINDINGS ................................................................................................ 16 

1 0.1 MORTALITY I CLINICAL SIGNS ......................................................................... 16 

10.2 BODY WEIGHTS ................................................................................................. 17 

10.3 MACROSCOPIC FINDINGS ................................................................................ 18 

11 GLP - CERTIFICATION ............................................................................................... 19 
1 PREFACE 

1.1 GENERAL 

Title 

Sponsor 

Study Monitor 

Test Facility 

Report 

Acute Oral Toxicity Study in Rats 

-

RCC Ltd 

Wolferstrasse 4 

CH-4414 FOilinsdorf I Switzerland 

1.2 RESPONSIBILITIES 

Study Director G. Arcelin 

Deputy for Study.Dir.ector._ . - --Dr:.-2.-Giannini .... 

Technical Coordinators 

Head ofRCC 

Quality Assurance 

1.3 SCHEDULE 

F. Frickert I M. Bernstein 

1. ·wothrich 

Experimental Starting Date 22-NOV-2005 

Experimental Completion Date 30-DEC-2005 

Delivery of Animals 22-NOV-2005 (females, 2000 mglkg) 

24-NOV-2005 (females, 2000 mglkg) 

07-DEC-2005 (females, 300 mglkg) 

09-DEC-2005 (females, 300 mglkg) 

Acclimatization 

Treatment 

22-NOV-2005 to 28-NOV-2005 (females, 2000 mglkg) 

24-NOV-2005 to 30-NOV-2005 (females, 2000 mglkg) 

07-DEC-2005 to 13-DEC-2005 (females, 300 mglkg) 

09-DEC-2005 to 15-DEC-2005 (females, 300 mglkg) 

29-NOV-2005 (females, 2000 mglkg) 

01-DEC-2005 (females, 2000 mglkg) 

14-0EC-2005 (females, 300 mglkg) 

16-DEC-2005 (females, 300 mg/kg) 

Page 3 
RCC STUDY NUMBERObservation 

Study Completion Date 

1.4 ARCHIVING 

Report 

22-NOV-2005 to 13-DEC-2005 (females, 2000 mg/kg) 

24-NOV-2005 to 15-DEC-2005 (females, 2000 mg/kg) 

07-DEC-2005 to 28-DEC-2005 (females, 300 mg/kg) 

09-DEC-2005 to 30-DEC-2005 (females, 300 mglkg) 

Page4 

RCC Ltd (CH-4452 ltingen I Switzerland) will retain the study plan, study plan amendment, 

raw data, a sample of test item(s) and the final report of the present study for at 1east ten 

years. No data will be discarded without the Sponsor's written consent. 
Report PageS 

1.5 SIGNATURE PAGE 

Study Director: 

Management: ~) Dr. H. Fankhauser 

date: Ol-{;r;..T -20()7 
Report 

1.6 QUALITY ASSURANCE GLP TOXICOLOGY 

RCC Ltd, Toxicology, CH-44521tingen I Switzerland 

STATEMENT 

RCC STUDY NUMBER : 

TEST ITEM 

STUDY DIRECTOR 

TITLE 

A40230 

G. Arcelin 

Acute Oral Toxicity Study in Rats 

PageS 

The general facilities and activitie.s are inspected periodically and the results are reported to 

the responsible person and the management. 

Study procedures, with exception of the fonnulation trials, were periodically audited. The 

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

below. 

Dates and Types of Dates of Reports to the Study Director 

QA Inspections and Test Facility Management 

17 -OCT -2005 Study Plan 17 -OCT -2005 

29-NOV-2005 Test System, Test Item, 29-NOV-2005 

Treatment, Dose Preparation, Raw Data 

13-JAN-2006 Draft Report 13-JAN-2006 

08-0CT -2007 Report 08-0CT -2007 

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

Quality Assurance: L. Michel 

C-n· uL____t; . . ...................... . date: 05- 0C.X - 2 ex:.:;::) 
Report 

GOOD LABORATORY PRACTICE 

1.7 STATEMENT OF COMPLIANCE 

RCC STUDY NUMBER : 

TEST ITEM 

STUDY DIRECTOR 

TITLE 

A40230 

G. Arcelin 

Acute Oral Toxicity Study in Rats 

Page7 

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 r~ating to Good 

Laboratory Practice, adopted May 1811:1, 2005 [RS 813.112.1]. 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)1861Final]. 

Study Director: 

~[fl.~ · date: ()3 J OCI v ~~ 
Report Page a 

1.8 TEST GUIDELINES 

The study procedures described in this report meet or exceed the requirements of the following guidelines: · 

Directive 2004n3/EC, 8.1 tris "Acute Oral Toxicity-Acute Toxic Class Method~ . April 29, 

2004. . 

OECD Guidelines for the Testini of Chemicats, Number 423 "Acute Oral Toxicity- Acute 

Toxic Class Method", adopted 17· December 2001 . 

1.9 ANIMAL WELFARE 

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

Animal Protection Law under license no. 34. 

1.10 SUMMARY OF STUDY PLAN AMENDMENT 

Study Plan Amendment No. 1: 

Change of study director and-deputy for study director. 
Report Page 9 

2 SUMMARY 

~oups, each of three female HanRcc:WIST {SPF) rats, were treated 

--by oral gavage administration at a dosage of 2000 mg/kg or 300 mg/kg body weight. 

The test item was diluted in vehicle (purified water) at a concentration of 0.2 g/ml or 

0.03 g/ml and administered at a volume dosage of 10 mUkg. 

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

and clinical signs were recorded. All animals were examined for clinical signs at approximately 30 minutes, 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test 

days 2-15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours 

after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. 

Body weights were recorded on ·day 1 (prior to administration) and on days 8 and 15. All 

animals were necropsied and examined macroscopically. 

The following animals were treated and percentage of mortality was observed: 

6 females treated at 2000 mg/kg 50 % 

6 females treated at 300 mg/kg 0 % 

Three animals treated at 2000 mg/kg were found dead five hours after application or <>n test 

day 2 or 3, respe9tiv~y . t.l~~in:'als treated at 300 mg/kg survived until the end of the study 

period. · 

Slightly to moderately ruffled fur was noted in three animals treated at 2000 mg/kg at the 1-

or 2-hour reading and persisted until their spontaneous death. In the remaining animals 

treated at this dosage slightly ruffled fur was observed from the 2- or 3-hour reading to the 

examination on test day 6, 8 or 9. Hunched posture was noted in three animals treated at 

2000 mg/kg from the 2- to 3-hour reading or at the observation five hours after treatment 

and again in one of these animals from test day 3 to 6. Two animals showed hunched 

posture only on test day 2 or 3. Feces containing mucus was seen in four animals at the 

observation on test day 2 and Persisted in one animal until test day 4. Soft fe.ces was 

observed in three animals on test day 3 or 5. 

No clinical signs were observed during the course of the study in all animals treated at 300 

mg/kg. 

A decrease in body weight (12.2%) was noted in one animal treated at 2000 mglkg within 

the first week after treatment, but recovered by the end of the study. The body weight of .the 

remaining animals was within the range commonly recorded for this strain and age. 

In two animals, which died spontaneously, liquid contents were recorded in the duodenum, 

ileum and jeunum. One of these two animals had a stomach distended with gas and a 

caecum with liquid content was noted, whereas in the second animal liquid contents were 

noted in the stomach. In the third animal a distended stomach was noted at the unscheduled 

necropsy. No macroscopic findings were recorded at necropsy of the remaining animals. 
3 CONCLUSION 

The median lethal dose 

rats, observed over a period 

Report Page 10 

after single oral administration to female 

300 mg/kg body weight < LDso (female rat) < 2000 mg/kg body weight 
Report Page 11 

4 PURPOSE 

The purpose of this study was to assess the acute toxicity 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 

Rationale 

Source 

Rat, HanRcc:WIST {SPF} 

Recognized by the international guidelines as a re<:ommended test system. 

RCC Ltd, Laboratory Animal Services 

CH-4414 FOIIinsdorf I Switzerland 

Number of animals per group 3 females 

Total number of animals 

Age when treated 

Identification 

Randomization 

Acclimatization 

5.2 HUSBANDRY 

Room no. 

Conditions 

Accommodation 

12 females 

12-13 weeks 

Unique -cage number and corresponding color-coded spots 

on the tail. The animals were marked at acclimatization start. 

Selected by hand at time of delivery. 

No eomputer generated randomization program. 

Under laboratory conditions, after health examination. Only 

animals without any visible signs of illness were used for the 

study. 

0105/ RCC Ltd, Fullinsdorf 

Standard Laboratory Conditions. Air -conditioned with 10-15 

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

relative humidity between 30-70% (values above 70% 

during cleaning process possible}, automatically controlled 

light cycle of 12 hours light and 12 hours dark, music during 

the daytime light period. 

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

tops and standard softwood bedding ('Lignocel' Schill AG, 

CH-4132 Muttenz/Switzerland}. 
Report Page 12 

Diet Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet, batch no. 39/05 (Provimi Kliba AG, CH-4303 

Kaiseraugst/Switzerland) ad libitum. Results of analyses for 

contaminants are archived at RCC Ltd. 

Water Community tap water from FOIIinsdorf ad libitum. Results of 

bacteriological, chemical and contaminant analyses are archived at RCC Ltd. 

5.3 TEST ITEM 

The following information was provide~ by the sponsor: 

Identification 

Description 

Batch number 

Purity 

Stability of test item 

Expiry date 

Stability of test item dilution 

Storage conditions 

Safety precautions 

5.4 VEHICLE 

Brown solid 

lab sample 

98% 

Stable under storage conditions. 

October 2008 

Stable in purified water for at least 30 day in the refrigerator. 

At room temperature (range of 20 ± 5 °C), light protected. 

Routine hygienic procedures were used to ensure the health 

and safety of the personnel. 

The following information was provided by RCC Ltd: 

Purified water prepared at RCC Ltd (deionised water which was processed and treated by 

the PURELAB Option-R unit. This latter links four purification technologies: reverse osmosis, 

adsorption, ion-exchange and photo oxidation). 

Purified water 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 formulation trial is excluded from the GLP statement of comp1iance. 
Report Page 13 

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 and a spatula as homogenizers. 

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 mglkg or 300 mglkg body weight after being fasted for approximately 17 to 19 hours 

(access to water was permitted). Food was provided again approximately 3 hours after 

dosing. 

The application volume was 10 mUkg body weight. 

Rationale: Oral administration wa·s 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 

6 PATHOLOGY 

6.1 NECROPSY 

Daily during the acclimatization period, during the first 

30 minutes and at approximately 1, 2, 3 and 5 hours after 

administration on test day 1 (with the clinical signs) and 

twice daily during days 2-15. 

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

Daily during the acclimatization period, during the first 

30 minutes and at approximately 1, 2, 3 and 5 hours after 

administration on test day 1. Once daily during days 2-15. All 

abnormalities were recorded. 

All animals which died spontaneously during the observat~on period were necropsied as 

soon as they were found dead. 

All surviving animals were killed· at the end of the observation period by Carbon dioxide 

asphyxiation and discarded after macroscopic examinations were performed. No organs or 

tissues were retained. 
Report Page 14 

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 Tox Computer System during recording and/or 

recorded on data sheets. · 

Macroscopic findings were compiled into the RCC Tox Computer System during recording. 

The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data 

collection, storage and retrievability. 
Report Page 15 

9 RESULTS 

9.1 MORTALITY 

Three animals treated at 2000 mg/kg were found dead five hours after application or on test 

day 2 or 3, respectively. All animals treated at 300 mg/kg survived until the end of the study 

period. 

9.2 CLINICAL SIGNS · 

Slightly to moderately ruffled fur was noted in three animals treated at 2000 mg/kg at the 1-

or 2-hour reading and persisted until their spontaneous death. In the remaining animals 

treated at this dosage slightly ruffled fur was observed from the 2- or 3-hour reading to the 

examination on test day 6, 8 or 9. Hunched .posture was noted in three animals treated at 

2000 mg/kg from the 2- to 3-hour reading or at the observation five hours after treatment 

and again in one of these animals from test day 3 to 6. Two animals showed hunched 

posture only on test day 2 or 3. Feces containing mucus was seen in four animals at the 

observation on test day 2 and persisted in one animal until test day 4. Soft feces was 

observed in three animals on test day 3 or 5. 

No clinical signs were observed during the course of the study in all animals treated at 300 

mg/kg. 

9.3 BODY WEIGHTS 

A decrease in body weight (12.2%} was noted in one animal treated at 2000 mg/kg within 

the first week after treatment, but recovered by the end of the study. The body weight of the 

remaining animals was within the range commonly recorded for this strain and age. 

9.4 MACROSCOPIC FINDINGS 

In two animals, which died spontaneously, liquid contents were recorded in the duodenum. 

ileum and jeunum. One of these two animals had a stomach distended with gas and a 

caecum with liquid content was noted, whereas in the second animal iiquid contents were 

noted in the stomach. In the third ·animal a distended stomach was noted at the unscheduled 

necropsy. No macroscopic findings were recorded at necropsy of the remaining animals. 

9.5 MEDIAN LETHAL DOSE 

The median lethal dose of after single oral administration to female 

rats, observed over a period of 14 days is: 

300 mg/kg body weight < LDso (female rat) < 2000 mg/kg body weight 
Report Page 16 

10 INDIVIDUAL FINDINGS 

10.1 MORTALITY I CLINICAL SIGNS 

Dose Ani- Test days 

mg/kg mal Sex Signs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

bw No. 0.5* 1* 2* 3* 5* 

2000 1 F No clinical signs ..j ..j ..j ..J ..J ..J ..J ..J ..J ..J 

Ruffled fur 1 1 1 1 1 1 1 1 1 

Hunched posture ..j 

Soft feces ..J 

Feces containing 

mucus ..J 

2 F No clinical signs ..J ..j ..J ..J ..J ..J ..J ..j ..J ..J ..J 

Ruffled fur 1 1 1 1 1 1 1 1 

Soft feces ..J 

Feces containing 

mucus ..J 

3 F No clinical signs ..J + 

Ruffled fur 1 1 2 

Hunched posture ..j ..j 

2000 4 F No clinical signs ..j ..j + 

Ruffled fur 1 1 1 2 

Hunched posture ..j 

Feces containing ..j 

mucus 

5 F No clinical signs ..J ..J ..J ..J ..J ..J ..j ..J 

Ruffled fur 1 1 1 1 1 1 1 1 1 1 1 

Hunched posture ..J ..J ..J ..J ..j 

Soft feces ..J 

Feces containing ..J ..J ..j 

mucus 

6 F No clinical signs ..J ..j + 

Ruffled fur 1 1 1 

Hunched posture ..j 

Ke: y 1 sli 9 ht, 2 moderate, + found dead, ..J noted 

• Examinations were performed approximately 0.5, 1, 2, 3 and 5 hours after treatment. 

No clinical signs were evident in any animal during the acclimatization period. 
Report Page 17 

Dose Ani- Test days 

mg/kg mal Sex Signs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

bw No. 0.5* 1* 2* 3* 5* 

300 7 F No clinical signs 

8 F No clinical signs " " " " " " " " " " " " " " " " " " " 

9 F No clinical signs " " " " " " " " " " " " " " " " " " " 

300 10 F No clinical signs " " " " " " " " " " " " " " " " " " " 

11 F No clinical signs " " " " " " " " " " " " " " " " " " " 

12 F No clinical signs " " " " " " " " " " " " " " " " " " " 

Key: ..J noted " " " " " " " " " " " " " " " " " " " 

* Examinations were performed approximately 0.5, 1, 2, 3 and 5 hours after treatment. 

No clinical signs were evident in any animal during the acclimat~ation period. 

10.2 BODY WEIGHTS 

Dose Animal Sex Day 1 DayS Day 15 

mg/kg No. I (treatment) 

2000 1 F 187.5 164.7 203.4 

2 F 184.1 208.4 222.5 

' 3 F 184.5 - - 2000 4 F 182.9 - -

5 F 187.2 189.6 214.6 

6 F 198.9 - - 300 7 F 180.5 197.5 205.7 

8 F 184.8 196.2 203.4 

9 F 186.4 206.4 211 .6 

300 10 F 180.1 199.4 200.7 

11 F 171.9 188.6 197.7 

12 F 191.5 215.2 223.2 

Body weights are presented in grams. 
Report Page 18 

10.3 MACROSCOPIC FINDINGS 

Dose Animal Sex Mode of Findings 

mg/kg No. death 

2000 1 F s No macroscopic findings 

2 F s No macroscopic findings 

3 F D Liquid contents in stomach, jejunum, ileum and duodenum 

2000 4 F D Distended stomach 

5 F s No macroscopic findings 

6 F D Liquid contents in jejunum, ileum, duodenum and caecum; 

stomach distended with gas 

300 7 F ·s No macroscopic findings 

8 F s No macroscopic findings 

9 F s No macroscopic findings 

300 10 F s No macroscopic findings 

11 F s No macroscopic findings 

12 F s No macroscopic findings 

S: scheduled necropsy, D: found dead 
Report 

11 GLP- CERTIFICATION 

The Swias GLP Monitoring Authorities 

Swi11F~I 

OIIJc:e ol 

PublicHMfth 

Statement of GLP Compliance 

It ia hereby confirmed that 

during lhe period of 

the following Fac:IHtiea of 

April 22, 25 - 29, 2005 

May 09-13, 2005 

RCC Ltd 

4452 ltlngen 

Switzerland 

were inspected by the Federal Offi~ of Public Health, the Swias Agency for Therapeutic 

Products and the Swias Agency for the Environment, Forests and Landscape with respect to 

lhe compliance with the Swisa legislation on Good Laboratory Practi~. 

Faellitfea 

• Teat Facility: Toxicology 

• Teat Facility: Environmental Chemlttry 

& Pharmanalytlca 

• Archive Facilities 

A,.a• of expertise • 

TOX, ACC, OTH (Safety 

Pharmacology, Alternative Test 

SY$tema) 

ACC, ECT, ENF, EMN, PCT, 

RES, OTH (Animal Metabolism) 

The inapeetion. were perfonned In agreement with the OECO Guidelines for National GlP 

Inspections and Audita. It was found that the aforementioned test facilities were opetatlng in 

complianoe with the Swi .. Ordinance relatln11 to Goo4 Laboratory PNctlce [RS 813.016.5] at 

the time they were inspected. 

Bem, November 2005 

Federal Office of Public Health 

The Director 

Prof. Th. Zeltner 

• TOX • Toxicolog)' ; 1\CO • Alllfytai iWICI Clinical Cll«nlllrt: ECT • E.....,_nlai iOIIidly en ~u.otle end '*'"trial 

organlltn• ; ENF • lleblviour ill....., • .oil-air. ~ion; EMN • Sludles on _... on _,. and natur11 ~; PCT • Plt~l testing: RES • Residue--. : OTH • OINr.IO lie .,.clfled. 

Page 19