## Acute Oral Toxicity Study in Rats Report 

## Author: G. Arcelin Sponsor: Olof Beckmann

Page 1 of 19Report 

### 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 ofAnimals 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 3RCC 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()7Report 

## 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 1 7-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 ofthe OECD Council (C(97)1861Final]. Study Director:

# ~[fl.~**·** date: 

## ()3 J 

## OCIv

### ~~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~~i n:'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 1 0

after single oral administration to female 

### 300 mg/kg body weight

### < 

## LDso (female rat) 

### < 2000 mg/kg body weightReport 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 oftest item Expiry date Stability oftest 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 ofthe 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 ofthe 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 record ing 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 retrievabil ity.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, butrecovered 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 ofthe 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 

### weightReport 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 ..J mucus 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 ..J mucus 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: 1 sli ht, 2 moderate, + found dead, ..J noted 

### y 9 • 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 

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# " " " 300 10 F No clinical signs 

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# " " 11 F No clinical signs 

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# " 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 deadReport 

## 11 GLP- CERTIFICATION The Swias GLP Mon itoring 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• ofexpertise • 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. Itwas 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 iWICIClinicalCll«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