TITLE PAGE 

FINAL REPORT

Acute oral toxicity study in rats 

TEST GUIDELINE(S)

OECD 423 

Commission Regulation (EC) No 440/2008 

US EPA OPPTS 870.1100 

Japan MAFF 8147 

STUDY COMPLETION DATE

April 17 2013 

SPONSOR

TEST FACILITY

AUTHOR(S)

TEST FACILITY PROJECT IDENTIFICATION

Project No.: 13-BF-OT010 

 PROJECT IDENTIFICATION

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PAGETLE 

PAGE 

This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This document is the 

property of the sponsor and may be used only for that purpose intended by the sponsor and as authorized under 

applicable law. Every other or additional use, exploitation, reproduction, publication or submission to other parties 

requires the written permission of the sponsor, except as otherwise required by applicable law (e.g., TSCA). The use of 

these studies/documents for regulatory, registration or other commercial purposes [including but not limited to 

Registration, Evaluation, Authorization and Restriction of Chemical Substances (REACH)], without the express 

written permission of the sponsor is unauthorized and strictly prohibited.
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THIS PAGE IS INTENTIONALLY LEFT BLANK 
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GLP COMPLIANCE STATEMENT 

This study was conducted in accordance with the OECD Principles of Good Laboratory 

Practice and the GLP Principles of the German "Chemikaliengesetz" in the current 

edition (Chemicals Act), which meet the United States Environmental Protection 

Agency Good Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part 792 

(TSCA)], with the exception that recognized differences exist between the GLP 

Principles/Standards of OECD and the Principles/Standards of FIFRA and TSCA. 

Typed name of Study Director:

Typed name of Laboratory: 

To be filled for USA EPA submission only: 

Sponsor: _______ Date: 

Typed name of Sponsor: 

Typed Name of Company: 

Submitter: _______ Date: 

Typed name of Submitter: 

Typed Name of Company: 
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The following statement is for US EPA submissions only: 

FLAGGING CRITERIA 

I have applied the criteria of 40 CFR 158.34/40 CFR161.34 for flagging studies for 

potential adverse effects to the results of the attached study. This study 

neither meets nor exceeds any of the applicable criteria. 

meets or exceeds the criteria numbered X.

Submitter: _______ Date: 

Typed Name of Signer: 

Typed Name of Company: 

Statement for US EPA submissions only: 

FLAGGING CRITERIA 

The flagging criteria of 40 CFR 158.34/40 CFR161.34 are not applicable for this study 

type. 
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STATEMENT OF THE QUALITY ASSURANCE UNIT 

Title of the report: 

 Acute oral toxicity study in rats 

Study director: 

Date of the final report: April 17, 2013 

The study was inspected regularly in accordance with the principles of Good Laboratory 

Practice for the testing of chemicals as specified by national (ChemG, in its current 

version) and international (OECD Principles on GLP as revised in 1997 

(ENV/MC/CHEM(98)17) standards. 

The conduct of this study was inspected at intervals to assure the integrity of the study. 

Inspections were performed according to the testing facility’s Quality Assurance 

program (i.e., the standard operating procedures of the Quality Assurance Unit). The 

final report was audited in detail against the approved study protocol and all pertinent 

raw data. 

The dates of all study inspections and of the final report as well the dates on which the 

respective findings were reported to management and to the study director are shown 

below. 

Phase of study Inspections Reports 

Study plan: February 25, 2013 February 25, 2013 

Inlife phase March 19, 2013 March 19, 2013 

Amendment to the plan: - - 

Study report: March 27, 2013 March 27, 2013 
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GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY)
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SIGNATURE PAGE

Study Director: 
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CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL 

Head of / 

Management: 

Study director: 

Pathology: 

Quality Assurance Unit (QAU): 

Acute Toxicology (Conduction): 
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CONTENTS 

TITLE PAGE 1

THIS PAGE IS INTENTIONALLY LEFT BLANK 2

GLP COMPLIANCE STATEMENT 3

STATEMENT OF THE QUALITY ASSURANCE UNIT 5

CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL 8

CONTENTS 9

1. SUMMARY 11

2. INTRODUCTION 12

2.1. OBJECTIVES 12

2.2. SELECTION OF DOSES/CONCENTRATIONS 12

2.3. TEST GUIDELINES 12

2.4. TIME SCHEDULE 13

2.5. RETENTION OF RECORDS 13

2.6. ANIMAL WELFARE 13

3. MATERIALS AND METHODS 14

3.1. TEST ITEM 14

3.2. TEST SYSTEMS 14

3.3. HOUSING AND DIET 15

3.4. TEST GROUPS AND DOSES/CONCENTRATIONS 16

3.5. TEST ITEM PREPARATION 16

3.6. ANALYSES 16

3.7. EXPERIMENTAL PROCEDURE 17

3.8. EVALUATION OF RESULTS 18

4. RESULTS 18

4.1. ANALYSES 18
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4.2. CLINICAL OBSERVATIONS 18

4.2.1. Mortality 18

4.2.2. Clinical signs 18

4.2.3. Body weights 19

4.3. PATHOLOGY 19

5. CONCLUSION 19

APPENDIX I 20

MORTALITY 21

MAXIMUM INCIDENCE OF CLINICAL SIGNS 21

BODY WEIGHTS CHANGES 22

GROSS PATHOLOGY 22

APPENDIX II 23

LIST OF ABBREVIATIONS 24
Page 11 of 24

1. SUMMARY

In an acute oral toxicity study performed according to the Acute Toxic Class method, 

2000 mg/kg bw of the test item (preparation in corn oil Ph.Eur.) 

were administered to two test groups of three fasted Wistar rats by gavage. 

The following test substance-related clinical observations were recorded on the day of 

application only: 

2000 mg/kg (first test group): 

• No mortality occurred

• Impaired general state in all animals

• Piloerection in all animals

• Cowering position in all animals

• Diarrhea in one out of three animals

2000 mg/kg (second test group): 

• No mortality occurred

• Impaired general state in one out of three animals

• Piloerection in one out of three animals

The mean body weight increased within the normal range throughout the study period. 

(with exception of one animal: the weight stagnated in the second observation week). 

There were no macroscopic pathological findings in the animals sacrificed at the end of 

the observation period. 

The acute oral LD50 was calculated to be 

LD50, oral, rat > 2000 mg/kg bw
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2. INTRODUCTION

2.1. OBJECTIVES 

The objective of this study was to assess the acute oral median lethal dose following a 

single oral application to female Wistar rats followed by a 14 day observation period. 

The study was performed in rats, because this species has been shown to be suitable 

for this type of study and is recommended by the test guidelines. 

2.2. SELECTION OF DOSES/CONCENTRATIONS 

By request of the sponsor a starting dose of 2000 mg/kg bw was chosen in the first step 

with 3 female animals. 

Because no mortality occurred, 2000 mg/kg bw were administered to another group of 

3 female animals in the second step. 

2.3. TEST GUIDELINES 

The study was conducted according to the following test guidelines: 

- OECD Guideline for Testing of Chemicals No. 423, December 17, 2001 (“Acute Oral 

Toxicity – Acute Toxic Class Method”) 

- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test 

methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorization and Restriction of 

Chemicals (REACH), Part B: Methods for the determination of toxicity and other 

health effects: Acute Oral Toxicity – Acute Toxic Class Method; Official Journal of 

the European Union, No. L 142 

- U. S. EPA Health Effects Test Guidelines OPPTS 870.1100, December 2002 

(“Acute Oral Toxicity”) 

- Japan MAFF Testing Guideline of 12 Nosan No. 8147 as this in line with OECD 423. 
Page 13 of 24

2.4. TIME SCHEDULE 

Test dates: Study initiation date 

(date of signature of the study plan) 

February 26, 2013 

Arrival of the animals and start of acclimatization 

period 

(at least 5 days before 

administration) 

Experimental starting date 

(selection of animals for the first treatment within 

a study) 

March 04, 2013 

First administration March 05, 2013 

Day of last observation March 26, 2013 

Experimental completion date 

(documentation of check of raw data) 

March 27, 2013 

2.5. RETENTION OF RECORDS 

The stability of the test item in the vehicle will be determined indirectly by concentration 

control analysis. For this purpose, the samples taken were subsequently deep-frozen 

and were sent to the sponsor for analysis. 

Additionally retained samples of the test item formulations were stored at at 

-20°C until finalization of the report and will be destroyed thereafter if no further 

analytical stability data are requested by the sponsor. At the end of the study (after 

finalization of the study report), GLP - relevant records and materials, the study plan 

and the final report will be shipped to the GLP archives of the sponsor: 

, 

and archived for at least the period of time specified in the GLP principles. 

Copies of raw data and the final report are archived by 

Details concerning responsibilities or locations of archiving can be 

seen from the respective SOPs of the above mentioned archiving locations. 

2.6. ANIMAL WELFARE 

This study was performed in accordance with the German Animal Welfare Act and the 

European Council Directive 86/609/EEC. 
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3. MATERIALS AND METHODS

3.1. TEST ITEM 

Analysis of the test item was carried out at . 

Information on stability or characterization of the test item, which appropriately defines 

the test batch, is under the responsibility of the sponsor. 

Name of test item: 

Test item No.: 12/0275-1 

Batch identification: 0008043725 

Purity: 93.8 area-% determined 

by GC with FID-analysis (for details see non-GLP 

analytical report No.: AU124464-1 provided by the 

sponsor.) 

Homogeneity: The test item was homogeneous by visual inspection. 

Storage stability: The stability of the test item under storage conditions 

over the test period was guaranteed by the sponsor, and 

the sponsor holds this responsibility. 

Storage conditions: Room temperature 

Physical state / color: Solid, melt / white 

Expiry date: December 2013 

3.2. TEST SYSTEMS 

Test species / strain / 

quality: 

Rat / Wistar / Crl:WI (Han) SPF 

Reasons for selection of the 

test species: 

Rats were selected since this rodent species is 

recommended in the respective test guidelines. 

Wistar rats were selected since there is extensive 

experience available in the laboratory with this strain of 

rats. 

Age on day 0: Young adult animals (female animals approx. 10 weeks) 
Page 15 of 24

Sex: As suggested by the OECD guideline nulliparous and 

non-pregnant female animals were used for the test, 

because there is no indication that male animals are 

likely to be more sensitive to the acute effects of the test 

item. 

Supplier: 

Arrival in the testing facility: Acclimatization period of at least 5 days before the 

beginning of the experimental phase; during the 

acclimatization period, the animals were accustomed to 

the environmental conditions of the study and to the diet. 

Identification: Individual identification by cage cards and tail marking. 

Body weight on day 0: Animals of comparable weight (± 20% of the mean 

weight, actual weights see Appendix I) 

3.3. HOUSING AND DIET 

Room temperature / 

relative humidity: 

The animals were housed in fully air-conditioned rooms. 

Central air-conditioning guaranteed a range of 22°C ±

3°C for temperature and of 30 – 70% for relative 

humidity. There were no deviations from these ranges, 

which influenced the results of the study. 

Air changes per hour Approx. 10 

Day / night rhythm: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

Type of cage: Makrolon cage, type III 

Number of animals per 

cage: Single housing 

Feeding: VRF1(P); SDS Special Diets Services, 67122 Altrip, 

Germany) 

Drinking water: Tap water ad libitum 

Bedding: H 15005-29; Ssniff, Spezialitäten GmbH (Experimental 

Animal Diets Inc., 59494 Soest, Germany) 

Enrichment: NGM E-022; ABEDD® LAB & VET Service GmbH, 

Hasnerstraße 84/6; 1160 Wien – Austria 
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3.4. TEST GROUPS AND DOSES/CONCENTRATIONS 

Dose 

(mg/kg bw)

2000 

2000 

Administration 

1 

2 

Administration volume

(mL/kg bw) 

10 

10 

Concentration 

(g/100 mL) 

20 

20 

No. of animals

(female) 

3 

3 

3.5. TEST ITEM PREPARATION 

Test item preparation and 

homogenization until end of 

each application period: 

The test item preparation was heated at approx. 60°C. 

The test item preparation was produced for each 

application group shortly before application by stirring 

with a magnetic stirrer. 

The preparation was administered lukewarm. 

Vehicle: Corn oil Ph.Eur. 

Reason for the selection of 

the vehicle: 

Solution in corn oil Ph.Eur. 

Form of application: Solution 

3.6. ANALYSES 

Conduct of analyses: A sample of the first concentration of the test item 

formulation was sent to the sponsor for analysis ( 

Analytical 

work will be done under the responsibility of the sponsor 

under GLP. The analytical report is not part of this study 

report. 

Stability of the test item 

preparation: 

The stability of the test item in the vehicle will be 

determined indirectly by concentration control analysis. 

For this purpose, the sample taken was stored at room 

temperature over the maximum duration of the 

administration period, subsequently deep-frozen and 

sent to the sponsor for analysis. 

Homogeneity of the test 

item preparation: 

The homogeneity of the test item preparation in the 

vehicle was not determined by analysis because the test 

item preparation was a solution. 
Page 17 of 24

Concentration control 

analysis of the test item 

preparation: 

A concentration control analysis of the test item 

preparation and archiving of the respective data will be 

done by the sponsor. 

Analysis of feed: The feed used in the study was assayed for chemical 

and microbial contaminants by the manufacturer in 

quarterly intervals. 

Analysis of drinking water: The drinking water was regularly assayed for 

contaminants by the municipal authorities of . 

The Drinking Water Regulation of Dec. 5, 1990 

served as the guideline for maximum tolerable 

contaminants. 

Bedding and enrichment 

analysis: 

The bedding and enrichment were regularly assayed for 

contaminants (chlorinated hydrocarbons and heavy 

metals). 

3.7. EXPERIMENTAL PROCEDURE 

Route of administration: Single oral administration by gavage. 

Fasting period: Feed was withdrawn from the animals at least 16 hours 

before administration, but water was available ad libitum. 

Time of day of 

administration: In the morning 

Observation period: 14 days 

Body weight determination: Individual body weights shortly before administration 

(day 0), weekly thereafter and on the last day of 

observation. 

Clinical observations: Recording of clinical signs several times on the day of 

administration, and at least once daily thereafter each 

workday for the individual animals. 

Mortality: A check for any dead or moribund animals was made at 

least once each workday, these records are archived by 
Page 18 of 24

Pathology: Necropsy with gross-pathology examination on the last 

day of the observation period after sacrifice by CO2-

inhalation in a chamber with increasing concentrations 

over time. 

Histology: No histological examinations were performed. 

3.8. EVALUATION OF RESULTS 

Calculations were performed using Microsoft Excel 2003 and checked with a calculator. 

4. RESULTS

Tables with the results of the clinical and pathological examinations are presented in 

the Appendix I. 

4.1. ANALYSES 

Analysis of the test item preparations will be done by the sponsor and reported 

separately. 

Analysis of feed: In view of the aim and duration of the study the 

contaminants occurring in commercial feed should not 

influence the results. 

Analysis of drinking water: In view of the aim and duration of the study there are no 

special requirements exceeding the specification of 

drinking water. 

Analysis of bedding and 

enrichment: 

In view of the aim and duration of the study there are no 

special requirements exceeding the specification of a 

commercial grade monitored by the manufacturer. 

4.2. CLINICAL OBSERVATIONS 

4.2.1. Mortality 

No mortality occurred in both 2000 mg/kg bw test groups. 

4.2.2. Clinical signs 

In all animals of the first 2000 mg/kg bw test group impaired general state and 

piloerection were observed at hour 1 and persisted in two animals until hour 4 or 5 after 

administration. 
Page 19 of 24

Diarrhea was noted in one animal at hour 0, while cowering position was observed in all 

animals at hour 1. 

Clinical signs in the second 2000 mg/kg bw test group revealed impaired general state 

and piloerection in one animal from hour 0 until hour 2 after administration. 

In two animals of this test group no clinical signs were observed during clinical 

examination. 

4.2.3. Body weights 

The mean body weight increased within the normal range throughout the study period. 

(with exception of one animal: the weight stagnated in the second observation week). 

4.3. PATHOLOGY 

There were no macroscopic pathological findings in the animals sacrificed at the end of 

the observation period. 

5. CONCLUSION

Under the conditions of this study the median lethal dose of after 

oral administration was found to be greater than 2000 mg/kg bw in rats. 
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APPENDIX I 
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MORTALITY 

MAXIMUM INCIDENCE OF CLINICAL SIGNS 
Page 22 of 24

BODY WEIGHTS CHANGES 

GROSS PATHOLOGY 
Page 23 of 24

APPENDIX II 
Page 24 of 24

LIST OF ABBREVIATIONS 

a.m. = ante meridiem (before noon) 

approx. = Approximately 

bw = body weight 

CMC = sodium Carboxymethylcellulose 

D, d = day(s) 

d0 = day 0 (day of application / day of administration) 

e.g. exempli gratia (for example) 

f = Female 

G; g = Gram 

h = Hour 

Kg; kg = Kilogram 

L = Liter 

m = Male 

mg = Milligram 

mL = Milliliter 

mm = Millimeter 

mw = Mean weight 

No.; no. = Number 

p.m. = post meridiem (after noon) 

SPF = specific pathogen free 

v = Volume 

v/v = volume per volume 

w = Weight 

WE = Week end/ public holydays 

w/w = weight per weight 

Symbols: 

°C = degree Celsius 

X = affected animals 

~ = Circa 

= = Analogous 

% = per cent 

- = no abnormality 

/ = no data 

+ = No further data 

 = Weight at death