Test Facility Study No. 

Page 1 of 113 

Study Title 

ACUTE INHALATION TOXICITY STUDY IN RATS

Test Guidelines 

OECD Guideline No. 403, Acute Inhalation Toxicity, May 1981 (REVISED 07 

September 2009); B2 of EEC Commission Directive, 92/69/EEC, July 1992 

(modified in 93/21/EEC, April 1993), US EPA Health Effects Test Guidelines, 

OPPTS 870.1300, Acute Inhalation Toxicity, August 1998 and US EPA for the 

toxicity, classification and label statements for inhalation exposure of pesticides 

(EPA737-B96-001). 

Author 

Study Completion Date 

16 July 2010 

Applicant/Sponsor 

Study Director/Test Facility 

Project Identification Numbers 

ID No.: 

Work Request No.: WR 

 Study No: (Unique Identifier) 
Test Facility Study No. 

2 

STATEMENTS OF CONFIDENTIALITY

The Following Statement Applies To Countries Other Than The United 

States of America: 

This report is the property of and contains 

confidential and trade secret information. Except as required by law, this report 

should not be partially or fully (i) photocopied or released in any form to an 

outside party without the prior written consent of and 

Company or its affiliates, or (ii) used by a registration authority to support the 

registration of any other product without the prior written consent of 

or its affiliates. 

The Following Statement Applies To The United States of America: 

STATEMENT OF NO DATA CONFIDENTIALITY

No claim of confidentiality is made for any information contained in this study on 

the basis of its falling within the scope of FIFRA Section 10(d) (l) (A), (B), or (C). 

Company 

Company Agent: 

__________________________________________________ 

U.S. Product Registration Manager 

_____________________ 

Date 



Test Facility Study No. 

6 

TABLE OF CONTENTS

Title Page ............................................................................................................1 

Statements of Confidentiality...............................................................................2 

Statement of No Data Confidentiality ..................................................................2 

Good Laboratory Practice Compliance Statement ................................................3 

Quality Assurance Documentation.......................................................................4 

Certification.........................................................................................................5 

Table of Contents.................................................................................................6 

List of General Abbreviations and Symbols .........................................................8 

Basic Study Information ......................................................................................9 

1.0 S ummary................................................................................................... 11 

2.0 In troduction............................................................................................... 13 

3.0 Materials and Methods .............................................................................. 14 

3.1 Te st Item............................................................................................... 14 

3.2 Animals and Management..................................................................... 14 

3.2.1 A nimal Supply ............................................................................. 14 

3.2.2 A cclimatisation ............................................................................ 15 

3.2.3 Allocation to Dose Group and Identification................................. 15 

3.2.4 Housing and Husbandry ............................................................... 15 

3.3 Tre atment ............................................................................................. 17 

3.3.1 D ose Groups ................................................................................ 17 

3.3.2 Justification of Exposure Level Selection ..................................... 17 

3.3.3 Route and Target Duration ........................................................... 17 

3.4 Inhalation Exposure System.................................................................. 17 

3.4.1 Pre-exposure Atmosphere Characterisation Investigations............ 17 

3.4.2 A tmosphere Generation................................................................ 17 

3.4.3 Exposure Chamber Design ........................................................... 17 

3.4.4 M ethod of Exposure..................................................................... 18 

3.4.5 Test Item Sampling and Analysis ................................................. 18 

3.5 In -life Observations .............................................................................. 18 

3.5.1 M ortality/Moribundity Checks .....................................................18 

3.5.2 Cl inical Observations ................................................................... 19 

3.5.3 B ody Weights .............................................................................. 19 

3.6 Te rminal Procedures............................................................................. 19 

3.6.1 Unscheduled and Scheduled Kill Investigations ........................... 19 

3.6.2 N ecropsy Procedures.................................................................... 19 

3.7 El ectronic Software............................................................................... 19 

3.8 S tatistical Analysis................................................................................ 20 

4 Deviations From Protocol.......................................................................... 20 

5 R esults....................................................................................................... 21 
Test Facility Study No. 

7 

5.1 Exposure Chamber Air Flow Rates, Temperature, Relative 

Humidity and Oxygen Levels................................................................ 21 

5.2 Exposure Chamber Atmosphere Conditions .......................................... 21 

5.2.1 Achieved Atmosphere Concentration ........................................... 21 

5.3 In -Life Observations ............................................................................. 22 

5.3.1 M ortality...................................................................................... 22 

5.3.2 Cl inical Observations ................................................................... 22 

5.3.3 B ody Weights .............................................................................. 23 

5.4 Te rminal Studies................................................................................... 24 

5.4.1 N ecropsy Examination ................................................................. 24 

5.5 L C50 Estimation .................................................................................... 24 

6.0 C onclusions............................................................................................... 24 

7.0 Retention of Records ................................................................................. 25 

TABLES

Table 1 Exposure Chamber Air Flow Rate, Temperature, Humidity 

and Oxygen Level ....................................................................... 26 

Table 2 Exposure Chamber Concentrations............................................... 30 

Table 3 Nominal Atmosphere Concentrations ........................................... 36 

Table 4 Mortality...................................................................................... 37 

Table 5 Clinical Observations ................................................................... 38 

Table 6 Body Weights (g)......................................................................... 53 

Table 7 Necropsy Findings ....................................................................... 57 

FIGURES

Figure 1 Schematic Diagram of Atmosphere Exposure System .................. 60 

APPENDICES

Appendix 1 GLP Documentation.................................................................... 61 

Appendix 2 Certificate of Analysis of Test Item............................................. 62 

Appendix 3 Individual Necropsy Findings...................................................... 63 

Appendix 4 Development and Validation of Methodologies for the 

Analysis of in Inhalation Toxicology Studies................ 79 
Test Facility Study No. 

8 

LIST OF GENERAL ABBREVIATIONS AND SYMBOLS

g gr am(s) 

GLP Good Laboratory Practice 

h h our(s) 

L l itre(s) 

m3 cubic metres 

mg m illigram(s) 

min mi nute(s) 

No. n umber 

SD s tandard deviation 

sec s econds 

°C degrees centigrade 

% per centage 

ppm parts per million 

GC gas chromatograph 
Test Facility Study No. 

9 

BASIC STUDY INFORMATION

Project Identification Numbers 

No: 

Study Title 

ACUTE INHALATION TOXICITY STUDY IN RATS 

Test Item 

Study Execution Dates 

Study Initiation Date: 05 February 2010 

Experimental Start Date: 09 February 2010 

Experimental Completion Date: 30 March 2010 

Study Completion Date 

16 July 2010 
Test Facility Study No. 

10 

Study Personnel and Test Facilities/Sites 

Senior Animal Unit Technician 

Inhalation Technologist 

Test Item Receipt and Dispense Under the Direction of

 

Necropsy Procedures Under the Direction of: 

Quality Assurance: 

Report Peer Review: 

Study Director

STUDY DIRECTOR TEST FACILITY ADDRESS

Sponsor Monitor

SPONSOR MONITOR COMPANY NAME ADDRESS
Test Facility Study No. 

11 

ACUTE INHALATION TOXICITY STUDY IN RATS

1.0 S UMMARY

The objective of the study was to determine the acute inhalation toxicity of the test 

item in rats following a single 4-h inhalation exposure. Animals were then 

retained for a 14 day post exposure observation period. 

Four groups of 5 male and 5 female Crl:CD®(SD IGS) rats were treated by noseonly inhalation exposure for up to 4 hours to gas in air. The test 

atmospheres were produced by quantitative dilution of the gas with air. Oxygen 

was added as required to maintain oxygen level. The concentrations of 

were sampled and measured by gas chromatography directly from the dosing 

chamber. Rats were exposed to target atmosphere concentrations of 125,000, 

50,000, 24,000 and 15,000 ppm of using a nose-only exposure 

technique. The following investigations were performed: clinical sign 

observation, body weight and macroscopic evaluations. 

Exposure Data 

Group mean achieved atmosphere concentrations of were 122,000, 

49,000, 25,400 and 14,600 ppm, respectively while group mean oxygen 

concentrations in the inhalation chamber were maintained between 20.3% and 

21.0%. 

Mortality 

There were a total of 13 deaths on this study. 

At 122,000 ppm exposure was terminated 20 minutes after the start of exposure, 

due to severe clinical signs and high mortality. Nine of 10 animals died during the 

exposure, with 1 animal being sacrificed for humane reasons at approximately 

20 minutes after exposure. 

At 49,000 ppm exposure was terminated approximately 4 minutes after the start, 

due to animal welfare considerations. One animal died within 3 minutes of the 

start of exposure and 1 died immediately after the 4 min exposure. The remaining 

8 animals exhibited severe clinical signs of toxicity and likely would have died if 

the exposure continued, but showed good recovery and were normal by 5 h after 

exposure. There were no further mortalities. 

At 25,400 ppm, 1 animal died after approximately 1 hour of exposure, the 

remaining animals survived the 4-h exposure. The surviving animals exhibited 

severe clinical signs of toxicity but at a lower incidence compared with higher 

concentration groups and showed recovery at approximately 24 h after exposure. 

There were no further mortalities. 

At 14,600 ppm all animals survived the 4-h exposure and showed no significant 

clinical signs. 
Test Facility Study No. 

12 

Clinical Observations 

A range of clinical observations related to treatment with included 

change in respiration pattern (fast, shallow, slow, laboured, gasping, cracking or 

irregular respiration/breathing), mainly noted during inhalation exposure and 

within the first few hours post exposure; pale appearance, red or brown staining 

around mouth and head, tail flicking, agitation, struggling, convulsing or 

moribund during inhalation exposure; subdued, hunched, piloerected, rolling gait, 

unkempt coat, convulsing, vocalizing, eye bulging, dull and dark eyes, salivation, 

red extremities, prostrated, walking on tip toes, partly closed eyes and tremors 

during the first few hours post exposure in surviving animals, mainly at 

concentrations of 122,000, 49,000 and 25,400 ppm. 

Body Weights 

No body weight observation was undertaken for animals exposed to 122,000 ppm 

due to early termination of the group. At 49,000 ppm, following 4 min inhalation 

exposure, 2 out of 3 surviving males showed a slight body weight reduction 

initially. The body weight gain was then normal. No abnormality was noted in 

female body weight profile. For animals exposed to 25,400 ppm for 4 h, an initial 

post exposure body weight loss was noted, but this was followed by satisfactory 

body weight gain throughout the remaining observation period. Body weight gain 

was satisfactory for animals exposed to 14,600 ppm. 

Necropsy Findings 

Reddened, dark, pale focus or spongy lungs were noted in many animals exposed 

to concentrations of 49,000 and 122,000 ppm, mainly in those animals that died or 

were killed prematurely. Speckled, dark or reddened thymus, enlarged 

mandibular lymph nodes and froth filled trachea were common findings in these 

animals. Dark or enlarged liver and dark pituitary gland was also commonly 

noted in these premature decedents. 

Speckled thymus, dark or enlarged liver and dark or reddened lungs were noted in 

isolated animals received concentrations at 25,400 ppm and 14,600 ppm, 

Conclusion 

Severe clinical signs and/or mortality were observed in animals exposed to 

concentrations of 122,000 and 49,000 ppm for 20 min and 4 min respectively. At 

25,400 ppm, there was 1 death during dosing, clinical signs of toxicity, and initial 

body weight loss post 4-h exposure. No adverse effects were observed in animals 

exposed to 14,600 ppm for 4 h. Based on the findings, the LC50 was estimated to 

be between 25,400 ppm and 49,000 ppm. 
Test Facility Study No. 

13 

2.0 I NTRODUCTION

The objective of the test was to determine the acute inhalation toxicity of 

in rats following a single 4-h inhalation exposure. Animals were then retained for 

a 14 day post exposure observation period. 

The rat was selected as the test model because of its ready availability and proven 

suitability in toxicology studies. The number of animals used was considered to 

be the smallest number necessary to provide sufficient data to meet the objectives 

of the study. The inhalation route of administration was selected because this 

route was defined as a possible route of human exposure. Nose-only exposure 

was used since this is the most applicable method of exposure for the test model. 

This study was designed to be compliant with OECD Guideline No. 403, Acute 

Inhalation Toxicity, May 1981 (REVISED 07 September 2009); B2 of EEC 

Commission Directive, 92/69/EEC, July 1992 (modified in 93/21/EEC, April 

1993) and US EPA Health Effects Test Guidelines, OPPTS 870.1300, Acute 

Inhalation Toxicity, August 1998. 

In addition, this study was designed to be compliant with the guidance provided 

by the US EPA for toxicity classification and label statements for inhalation 

exposure of pesticides (EPA737-B-96-001), the guide for the labelling of 

dangerous substances published in the Official Journal of European Communities 

(EEC Directive 93/21) and the guidance provided by the U.N. Globally 

Harmonised System of Classification and Labelling of Chemicals (2007). 

The study was conducted at 

The study was sponsored by

The study timings were as follows: 

Study Initiation Date: 05 February 2010 

Experimental Start Date: 09 February 2010 

Experimental Completion Date: 30 March 2010 

Study Completion Date: 16 July 2010 
Test Facility Study No. 

14 

3.0 M ATERIALS AND METHODS

3.1 Test Item 

The test item, was supplied as a liquefied gas in an appropriate pressure 

cylinder by the Sponsor and arrived at on 11 January 

2010. The test item was used as supplied. 

The Sponsor supplied a Certificate of Analysis for the test item along with a 

Material Safety Data Sheet. A copy of the test item Certificate of Analysis is 

presented in Appendix 2. 

Test Item: ( Number) 

Alternative Name: 

Batch/Lot No.: 

Description: Liquid under pressure in gas cylinder 

(BP+7.5°C) 

Purity: 99. 971 (mole %) 

Storage Conditions: Ambient, gas cylinder 

Expiry Date: 30 June 2011 

Supplier: 

3.2 Animals and Management 

3.2.1 A nimal Supply

A total of 20 male and 20 female rats (Crl:CD® (SD IGS)) were supplied by 

The first batch consisting of 10 male and 10 female animals arrived at 

 on 09 February 2010. Animals weighed approximately 200-

243g (males) and 151-175g (females). 

The second batch consisting of 5 male and 5 female animals arrived at 

 on 23 February 2010. Animals weighed approximately 200-

218g (males) and 153-167g (females). 

The third batch consisting of 5 males and 5 females animals arrived at 

on 09 March 2010. Animals weighed approximately 205-220g 

(males) and 185-197g (females). 

For all three batches females were nulliparous and non pregnant. All animals 

were approximately 6-7 weeks old on arrival 

On day of exposure, all Group 3 (Target concentration of 125,000 ppm) animals 

were approximately 7-8 weeks old and weighed approximately 253-307g and 181-

194g for males and females, respectively. 
Test Facility Study No. 

15 

On day of exposure, all Group 2 (Target concentration of 50,000 ppm) animals 

were approximately 8-9 weeks old and weighed approximately 273-343g and 193-

219g for males and females, respectively. 

On day of exposure, all Group1 (Target concentration of 24,000 ppm) animals 

were approximately 7-8 weeks old and weighed approximately 228-259g and 170-

189g for males and females, respectively. 

On day of exposure, all Group 4 (Target concentration of 15,000 ppm) animals 

were approximately 7-8 weeks old and weighed approximately 231-255g and 186-

217g for males and females, respectively. 

3.2.2 A cclimatisation

Within 24 h of arrival, all animals were given a clinical examination by an 

experienced animal technician. All animals received were deemed to be 

satisfactory for use in the study. 

All animals were allowed to acclimatise to the rodent 

toxicology accommodation (Block N) for at least 6 days before the 

commencement of dosing. For 5 days before commencement of dosing, the 

animals were conditioned to the restraint procedures used for nose-only exposure 

by placing the animals in restraint tubes for gradually increasing periods of time. 

On the first day, this took the form of an introduction and hold within the restraint 

tube. This procedure progressed thereafter from ca 30 min up to 4 h. 

3.2.3 Allocation to Dose Group and Identification

The animals were allocated to 4 dose groups. No formal randomisation procedure 

was applied. Animals were removed from their transport box and allocated on 

arrival by introduction to cages labelled with study number, animal number, sex, 

cage number and dose group. Males and females were housed separately. 

Each animal received an ‘ear notch’ which identified it individually within the 

study. 

3.2.4 Housing and Husbandry

The animals were housed in a separate animal holding room and moved to an 

adjacent laboratory for dosing. 

Cage type: Solid bottom polycarbonate/polypropylene cages 

with stainless steel mesh tops containing an integral 

food hopper with 1 to 2 plastic water bottles 

Cage size: Approximately 61 x 43.5 x 24 cm 

Bedding material: Wood shavings* 

Cage rack: Cages were suspended on movable racks 

Animal housing: 5 per cage by sex 

Environmental enrichment: Wooden chewsticks* 
Test Facility Study No. 

16 

* = wood shavings and wooden chewsticks were supplied with a certificate of 

analysis for significant contaminants. An analytical certificate of analysis for each 

batch used is retained in the archives at 

Cages and water bottles were changed as necessary throughout the course of the 

study. 

There was automatic control of temperature and humidity which was monitored 

and recorded daily. There was also automatic control of light cycle. 

Target room temperature: 18-22°C 

Target room relative humidity: 40-70% 

Room airflow: A minimum of 15 air changes per hour 

Light hours: No rmally 0700-1900 

Actual room temperature and relative humidity ranges were 15.90-27.19o

C and 

38.18-87.02% respectively. 

The animal room floor and work surfaces were washed daily with disinfectant 

solution (0.5% Tego 2000, Th. Goldschmidt and Company Limited, Middlesex, 

UK). Approximately once weekly, the walls, ceiling and all other surfaces within 

the animal room were washed with disinfectant solution. 

Food type: Rat and Mouse (modified) No. 1 Diet SQC Expanded* 

Food supplier: Special Diets Services, 1 Stepfield, Witham, Essex, UK 

Feeding regime: Available to the animals ad libitum, except during 

inhalation exposure, and other routine investigations. 

* = An analytical certificate for each batch of diet used is retained in the archives 

at 

The food used is considered not to contain any additional substances in sufficient 

concentration to have any influence on the outcome of the study. The food was 

supplied with a batch analysis for major nutritive components and significant 

contaminants and was used within the manufacturer’s designated shelf-life. 

Water type: Domestic mains drinking water* 

Water access: Available to the animals ad libitum, except during 

inhalation exposure, and other routine investigations. 

* = The water used by is analysed at 6 month intervals 

for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites. 

A bacteriological screen is also undertaken. An analytical certificate for all 

applicable analyses in relation to the conduct of the study is retained in the 

archives at 

The water used was considered not to contain any additional substances in 

sufficient concentration to have any influence on the outcome of the study. 
Test Facility Study No. 

17 

3.3 T reatment 

3.3.1 Dos e Groups

The animals were allocated to 4 dose groups and treated as follows: 

ANIMAL NUMBERS

DOSE GROUP

TARGET ATMOSPHERE 

CONCENTRATION 

(PPM) MALES FEMALES

3-High Dose 125,000 21-25 26-30 

2-Intermediate Dose 50,000 11-15 16-20 

1-Low Dose 24,000 1-5 6-10 

4-New Low Dose 15,000 31-35 36-40 

3.3.2 Justification of Exposure Level Selection

The initial animal exposure to test item was 125,000 ppm. Due to the number of 

deaths, exposures to lower concentrations were performed. 

3.3.3 Route and Target Duration

Inhalation method: Nose-only exposure 

Frequency: O nce 

Exposure duration: 4 h 

Treatment period: 1 day 

Post dose observation period: 14 days 

3.4 Inhalation Exposure System 

3.4.1 Pre-exposure Atmosphere Characterisation Investigations

Details of preliminary atmosphere characterisation investigations are retained 

within the study data. 

Satisfactory attainment of the test item exposure concentration was demonstrated 

prior to the commencement of the animal exposures. 

3.4.2 A tmosphere Generation

The chamber atmospheres were generated by quantitative dilution of the gas test 

item with air. When required, oxygen was added to maintain at least 19% oxygen 

in the test atmosphere. Concentrations were controlled by varying the volume of 

gas through an appropriate pressure regulator attached to the gas cylinder. 

3.4.3 Exposure Chamber Design

The exposure chamber was a modular, flow past design (See Figure 1) constructed 

from stainless steel. This design allowed continuous supply of test atmosphere to 

be delivered to each animal; the biased flow ensured that there was no rebreathing of the generated test atmosphere. 
Test Facility Study No. 

18 

The exposure chamber was located in an extract booth (for the protection of 

personnel) and operated to sustain a dynamic air flow sufficient to ensure an 

evenly distributed exposure atmosphere. All air flow rates (delivered and 

extracted) were monitored visually using calibrated flow meters. Chamber air 

flow rates, temperature and relative humidity levels were recorded at regular 

intervals (approximately every 30 min) during the exposure period. 

3.4.4 M ethod of Exposure

For inhalation exposure, the rats were restrained in clear, tapered, polycarbonate 

tubes with an adjustable back-stop to prevent the animals from turning in the 

tubes. The animals’ snouts protruded through the anterior end of the restraint 

tubes which were connected to the exposure chamber by way of push-fit through 

an inhalation port in the chamber wall. This exposure technique was used to 

minimise concurrent exposure by the oral and dermal routes. All animals were 

exposed on a single tier of the exposure chamber to eliminate any significant 

spatial exposure variation. 

3.4.5 Test Item Sampling and Analysis

Prior to and during exposure the concentration of the test gas was determined by 

gas chromatography. Chamber atmosphere was drawn from the breathing zone of 

the animals through an automated gas sampling loop and a fixed volume directly 

injected into a gas chromatograph. The gas samples were analysed using the 

method supplied by the Sponsor and validated at under 

 Study No. Method (Appendix 4) 

The nominal exposure chamber test atmosphere concentration (ppm) was 

calculated based on the weight of test item utilised during atmosphere generation 

and the total air volume that flowed through the exposure chamber, using the 

following equation. 

Nominal Concentration (ppm) = Weight of Test Item (mg) x 24.5 

 Total Air Volume (m3

) MW 

MW = molecular weight of = 

3.5 In -life Observations 

3.5.1 M ortality/Moribundity Checks

All animals were checked in the morning and as late as possible each day for 

viability. 
Test Facility Study No. 

19 

3.5.2 C linical Observations

All the animals were examined for reaction to treatment during the 4-h exposure 

period. Any clinical signs noted were recorded at target 30 min intervals with 

some supplementary signs recorded as they were noted and immediately post 

dose. Surviving animals were also observed approximately 1-2 h and up to 7h 

post exposure. The onset, intensity and duration of any signs observed were 

recorded and reported per individual animal. 

Observations included (but were not limited to) the following: 

 -changes in skin, fur, eyes and mucous membranes; 

 -effects in respiratory, circulatory, autonomic and central nervous system; 

 -effects on somato-motor activity and behaviour pattern; 

 -observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep 

and coma; 

During the pretrial and 14 day post exposure observation periods the animals were 

observed at least once daily. 

3.5.3 B ody Weights

Body weights were recorded once pretrial and immediately prior to 

commencement of exposure. Animals were also weighed on Days 1, 3, 7, 10 and 

14 of the post exposure observation period of the study. 

3.6 T erminal Procedures 

3.6.1 Unscheduled and Scheduled Kill Investigations

All surviving animals were killed and necropsied after completion of the 14 day 

post exposure observation period (Day 15 of the post exposure observation 

period). 

3.6.2 N ecropsy Procedures

All animals were killed by exposure to carbon dioxide and body weights recorded 

prior to necropsy. The necropsy consisted of an external examination and 

macroscopic examination of the contents of the thoracic and abdominal cavities. 

All gross abnormalities were, where appropriate, recorded in descriptive terms, 

including location(s), size (in mm), shape, colour, consistency and number. Any 

abnormalities were preserved in an appropriate preservative solution. The 

respiratory tract was closely examined for signs of gross irritation. 

On completion of the necropsy, the carcasses were disposed of. 

3.7 El ectronic Software 

Details pertaining to the software programs used are retained in the study data 

files and are not reported herein. 
Test Facility Study No. 

20 

3.8 Statis tical Analysis 

No formal statistical analysis was conducted. 

4 D EVIATIONS FROM PROTOCOL

The protocol required target ranges of room temperature and relative humidity 

were 18-22°C and 40-70% respectively. The actual room temperature and relative 

humidity ranges were 15.90-27.19°C and 38.18-87.02% respectively. 

Protocol required clinical observations to be undertaken at regular intervals during 

inhalation exposure, immediately post exposure and approximately 1-2 h post 

exposure. However the requirement was not always followed and more frequent 

observations were performed due to the responses of animals to the test item. 

Protocol required animals to be acclimatised to rodent 

toxicology accommodation for a period of at least 7 days before the 

commencement of dosing. Animals exposed to target concentration of 125,000 

ppm however were acclimatised for 6 days only. 

Animals at age of approximately 7-8 weeks and weight of approximatenly 

150-200 g for females and 200-250 g for males on commencement of exposure 

were required by the protocol. The actual age range was 7-9 weeks and weight 

range was 170-219 g for females and 228-343 g for males on commencement of 

exposure. 

Nominal concentration of test atmosphere was calculated and reported in surplus 

to protocol requirement. This was considered to be necessary to give an indication 

of generation efficiency. 

These deviations from protocol are considered not to have had any impact on the 

final outcome or integrity of the study. 
Test Facility Study No. 

21 

5 R ESULTS

5.1 Exposure Chamber Air Flow Rates, Temperature, Relative Humidity and 

Oxygen Levels 

The exposure chamber air flow rates, temperature, relative humidity levels and 

oxygen levels are presented in Table 1. 

Group mean values of exposure chamber temperature, relative humidity and 

oxygen levels are as follows: 

DOSE GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

TEMPERATUREA

(°C) 

RELATIVE HUMIDITYA

(%) 

O2A 

(%) 

3 

(125,000) 

25.0 

(0.3) 

B 20.4 

(0.4) 

2 

(50,000) 

24.8 B 20.7 

1 

(24,000) 

23.1 

(0.3) 

2.0 

(1.6) 

20.3 

(0.3) 

4 

(15,000) 

23.7 

(0.2) 

1.0 

(0.9) 

21.0 

(0.2) A = standard deviation values presented in parentheses. 

B

 = no valid result available for reporting due to suspect data recording error. 

Except for the relative humidity for Groups 2 and 3 which was suspected to have 

recording error, the temperature, relative humidity and oxygen levels were 

considered to be acceptable for this type of generation system. 

5.2 Exposure Chamber Atmosphere Conditions 

5.2.1 Achieved Atmosphere Concentration

Individual and mean atmosphere concentration measurements are presented in 

Table 2, whilst the nominal atmosphere concentrations are presented in Table 3. 
Test Facility Study No. 

22 

DOSE GROUP/TARGET 

ATMOSPHERE CONCENTRATION 

(PPM) 

ACHIEVED ATMOSPHERE 

CONCENTRATIONA 

(PPM) 

NOMINAL CONCENTRATION 

(PPM) 

3 

(125,000) 

121,979 

(2192) 127,703 

2 

(50,000) 

49,003 

(1715) 60,663 

1 

(24,000) 

25,406 

(963) 36,041 

4 

(15,000) 

14,642 

(391) 24,876 

A = standard deviation values presented in parentheses. 

The difference between the nominal and the measured atmosphere concentrations 

is considered typical when utilising the type of generation/exposure system and 

methodology used on this study. 

5.3 In -Life Observations 

5.3.1 Mor tality

There were a total of 13 unscheduled deaths on the study. Mortality results are 

presented in Table 4. 

At 122,000 ppm, 5 males and 4 females were found dead after 20 min inhalation 

dosing. One female was killed prematurely due to the severity of clinical signs. 

At 49,000 ppm, 2 males were found dead, one during the 4 min dosing and one 

immediately post dosing. The study was terminated due to severe clinical signs, 

and this level was presumed to result in 100% mortality. 

At 25,400 ppm, 1 male was found dead at approximately 1 h into inhalation 

dosing 

There was no mortality at 15,000 ppm. 

5.3.2 C linical Observations

A summary of clinical signs observed, their duration and the number of animals 

affected is presented in Table 5. 

At 122,000 ppm, during the 20 min inhalation dosing, animals were noted to have 

initially fast respiration with extreme agitation and tail flicking then at 

ca 20 minutes the tail flicking had stopped and the animals had very slow 

respiration, very pale appearance, red/brown staining around eyes, slight to 

marked red staining around mouth and unkempt coats. Before being killed 

prematurely, the only surviving animal had laboured and crackling respiration, 

subdued behaviour, piloerection, convulsions, vocalisation, bulging eyes and red 

liquid from mouth and eyes. 
Test Facility Study No. 

23 

At 49,000 ppm, within the 4 min inhalation exposure, all animals showed extreme 

agitation, tail flicking, gasping, convulsions and red liquid from mouth with one 

male additionally showing pale, prostrate and moribund. The study was stopped 

prematurely due to the severity of the clinical signs, with the presumption that 

continued exposure would produce 100% mortality. Surviving animals showed 

the signs of prostrate, pale, eyes dull, laboured breathing, red liquid from mouth, 

salivation, subdued behaviour, red staining around mouth, red extremities, 

gasping, red staining around eyes, irregular breathing and hunched within the first 

two hours post exposure. The three remaining males continued to show signs of 

red staining around mouth, red staining around eyes and hunched at three hours 

post exposure but no abnormal clinical signs were noted for all females. The 

males had returned to normal by the following day. 

At 25,400 ppm, animals showed tail flicking, struggling in tube, laboured, 

irregular and shallow breathing, gasping, pale skin, salivation, dark eyes and red 

liquid from mouth during the 4-h inhalation exposure, but with a lower incidence 

when compared with the animals in higher concentration groups. The signs of 

hunched posture, subdued behaviour and piloerection, rolling gait, eyes partially 

closed, and unkempt coat were commonly noted in surviving animals within the 4 

h post exposure observation period with a few animals showing staining around 

nose, eyes, head and thorax. Salivation was only noted immediately post dose for 

most animals. Walking on tip toes and tremors were observed in few animals 

later on in the period. Unkempt coat in a few females was the only sign noted 

within 3 days post exposure. 

At 14,600 ppm, the only clinical sign noted was tail flicking at the start of dosing 

during 4-h inhalation exposure. There was no abnormal clinical signs post 

exposure except for a lump on ventral abdomen in one female from 8th day post 

exposure which is considered to be unrelated to treatment with test item. 

5.3.3 B ody Weights

Individual and group mean body weight values are presented in Table 6. 

At 122,000 ppm, no body weight assessment was undertaken animals due to 

mortality. 

At 49,000 ppm, following 4 min inhalation exposure, 2 out of 3 surviving males 

showed a slight body weight reduction initially. The body weight gain was then 

normal. No abnormality was noted in female body weight profile. The study was 

stopped prematurely due to severe clinical signs, with the presumption that 

continued exposure would produce 100% mortality. 

At 25,400 ppm, animals had an initial body weight loss after 4-h inhalation 

exposure, followed by steady increase in body weight throughout the remaining 

post exposure observation period. 

At 14,600 ppm, a satisfactory increase in body weight during post exposure period 

was observed. 
Test Facility Study No. 

24 

5.4 T erminal Studies 

5.4.1 N ecropsy Examination

Necropsy findings are presented in Table 7 and individual necropsy findings are 

presented in Appendix 3. 

At 122,000 ppm, nearly all animals had reddened lungs except one male which 

had dark lungs. Pale foci were noted in one female and two males had spongy 

lungs with froth filled trachea. Reddened mandibular lymph nodes were noted in 

two females and two males; in one of the males these nodes were also enlarged. 

All but one animal showed a reddened, dark and/or speckled thymus. Dark or 

enlarged liver, some with pale foci and prominent lobulation was observed in two 

males and three females. Staining of the skin was noted in three males and two 

females, a further male had reddening of the skin. Dark pituitary gland was noted 

in four males and two females. A dark testis was noted in one male. A distended 

urinary bladder was noted in another male. One male and one female showed 

prominent intestines. One male had a cranium that contained blood. 

At 49,000 ppm, only two male animals exhibited the findings of dark lungs, froth 

filled trachea, speckled thymus and staining of skin and subcutis. Additionally one 

of these animals had a dark liver. The study was stopped prematurely due to 

severe clinical signs, with the presumption that continued exposure would produce 

100% mortality. 

At 25,400 ppm, the only finding was a speckled thymus in one male. 

At 14,600 ppm, one male had reddened lungs as well as an enlarged, dark liver 

and one female had dark lungs. 

5.5 L C50 Estimation 

Based on the findings, the LC50 was estimated to be between 25,400 ppm and 

49,000 ppm. 

6.0 C ONCLUSIONS

Severe clinical signs and/or mortality were observed in animals exposed to 

concentrations of 122,000 and 49,000 ppm for 20 min and 4 min respectively. At 

25,400 ppm, there was 1 death during dosing, clinical signs of toxicity, and initial 

body weight loss post 4-h exposure. No adverse effects were observed in animals 

exposed to 14,600 ppm for 4 h. Based on the findings, the LC50 was estimated to 

be between 25,400 ppm and 49,000 ppm. 
Test Facility Study No. 

25 

7.0 R ETENTION OF RECORDS

All raw data and pertinent information, including the original study plan, any 

amendments, and the final report will be archived at 

for 2 years. At the end of this period, these materials (except for the final 

report with original signatures) will be transferred to the Sponsor’s GLP facility in 

Facility/site-specific raw data (such as equipment logs, temperature logs and 

personnel records) will be maintained at the facility where the work was 

performed. 
Test Facility Study No. 

26 

TABLE 1 E XPOSURE CHAMBER AIR FLOW RATE, TEMPERATURE,

HUMIDITY AND OXYGEN LEVEL

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

TARGET TIME 

DURING 

EXPOSURE A 

(MIN) 

AIR FLOW 

(L/MIN) 

TEMPERATURE 

(

O

C) 

RELATIVE 

HUMIDITY 

(%) 

O2 

(%)

0 4. 917 24.8 - 20.1 

+20 4. 917 25.2 B 20.6 

Mean 4. 917 25.0 B 20. 4 

3 

(125,000) 

SD 0. 000 0.28 N/A 0.35 

- = humidity not recorded in error 

A

 = time relative to commencement of animal exposure 

B

 = no valid result available for reporting due to suspect data recording error. 

N/A = not applicable 

SD = standard deviation 
Test Facility Study No. 

27 

TABLE 1 E XPOSURE CHAMBER AIR FLOW RATE, TEMPERATURE,

HUMIDITY AND OXYGEN LEVEL (CONTINUED) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

TARGET TIME 

DURING 

EXPOSURE A 

(MIN) 

AIR FLOW 

(L/MIN) 

TEMPERATURE 

(

O

C) 

RELATIVE 

HUMIDITY 

(%) 

O2 

(%)

2 

(50,000) 0 5. 003 24.8 B 20.7 

A

 = time relative to commencement of animal exposure 

B

 = no valid result available for reporting due to suspect data recording error. 
Test Facility Study No. 

28 

TABLE 1 E XPOSURE CHAMBER AIR FLOW RATE, TEMPERATURE,

HUMIDITY AND OXYGEN LEVEL (CONTINUED) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

TARGET TIME 

DURING 

EXPOSURE A 

(MIN) 

AIR FLOW 

(L/MIN) 

TEMPERATURE 

(

O

C) 

RELATIVE 

HUMIDITY 

(%) 

O2 

(%)

0 5. 516 22.5 1.5 20.3 

+30 5. 516 23.7 1.9 20.2 

+60 5. 516 23.4 2.1 20.2 

+90 5. 516 22.9 0.9 20.1 

+120 5. 516 22.9 0.2 20.2 

+150 5. 516 23.1 6.2 20.2 

+180 5. 516 22.9 1.6 20.2 

+210 5. 516 23.2 1.7 20.2 

+240 5. 516 23.1 1.7 21.3 

+270 5. 516 23.1 2.5 20.3 

Mean 5. 516 23.1 2.0 20.3 

1 

(24,000) 

SD 0. 000 0. 3 1.6 0.3 

A

 = time relative to commencement of animal exposure 

SD = standard deviation 

 
Test Facility Study No. 

29 

TABLE 1 E XPOSURE CHAMBER AIR FLOW RATE, TEMPERATURE,

HUMIDITY AND OXYGEN LEVEL (CONTINUED) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

TARGET TIME 

DURING 

EXPOSURE A 

(MIN) 

AIR FLOW 

(L/MIN) 

TEMPERATURE 

(

O

C) 

RELATIVE 

HUMIDITY 

(%) 

O2 

(%)

0 4. 996 23.6 0.6 20.8 

+30 4. 996 23.6 0.4 20.8 

+60 4. 996 24.1 2.3 20.9 

+90 4. 996 23.6 2.9 20.8 

+120 4. 996 23.6 0.3 21.1 

+150 4. 996 23.6 0.8 21.0 

+180 4. 996 23.6 0.6 20.9 

+210 4. 996 23.7 0.6 21.4 

+240 4. 996 23.6 0.3 20.9 

Mean 4. 996 23.7 1.0 21.0 

4 

(15,000) 

SD 0. 000 0. 2 0.9 0.2 

A

 = time relative to commencement of animal exposure 

SD = standard deviation 
Test Facility Study No. 

30 

TABLE 2 E XPOSURE CHAMBER CONCENTRATIONS

Total Test Atmosphere Generation Period: 72 min 

Animal Exposure Time: 20 min 

Test Item Air Flow Rate: 0.674 L/min 

O2 Air Flow Rate: 0.253 L/min 

Dilution Air Flow Rate: 3.99 L/min 

DOSE 

GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

SAMPLE TIMEA

 

(MIN) SAMPLE CODE

EXPOSURE CHAMBER 

CONCENTRATIONB

(PPM) 

MEAN CHAMBER 

CONCENTRATIONC

(PPM) 

-4 Group 3 Test 4 124506 

18 Group 3 Sample 1 120592 

3 

(125,000)

21 Group 3 Post 1 120840 

121,979 

(2192) 

Animal exposure stopped at 20 min into dosing due to severe clinical signs and high mortality. 

Mean chamber concentration calculated from the samples taken during, immediately before and immediately after the 

inhalation exposure due to only one sample being taken during exposure. 

A

 = time relative to commencement of animal exposure. 

B

 = exposure chamber atmosphere of test item was sampled and measured using a gas chromatograph (GC) connected to the 

intake of the dosing chamber. 

C

 = standard deviation values presented in parentheses. 
Test Facility Study No. 

31 

TABLE 2 E XPOSURE CHAMBER CONCENTRATIONS (CONTINUED) 

Total Test Atmosphere Generation Period: 31 min 

Animal Exposure Time: 4 min 

Test Item Air Flow Rate: 0.252 L/min 

O2 Air Flow Rate: 0.073 L/min 

Dilution Air Flow Rate: 4.678 L/min 

DOSE 

GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

SAMPLE TIMEA

 

(MIN) SAMPLE CODE

EXPOSURE CHAMBER 

CONCENTRATIONB 

(PPM) 

MEAN CHAMBER 

CONCENTRATIONC 

(PPM) 

-7 Group 2 Test 3 48691 

0 Group 2 Sample 1 47465 

2 

(50,000)

12 Group 2 Post 1 50852 

49,003 

(1715)

Animal exposure stopped at 4 min into dosing due to severe clinical signs and mortality. 

Mean chamber concentration calculated from the samples taken during, immediately before and immediately after the 

inhalation exposure due to only one sample being taken during exposure. 

A

 = time relative to commencement of animal exposure. 

B

 = exposure chamber atmosphere of test item was sampled and measured using a gas chromatograph (GC) connected to the 

intake of the dosing chamber. 

C

 = standard deviation values presented in parentheses. 
Test Facility Study No. 

32 

TABLE 2 E XPOSURE CHAMBER CONCENTRATIONS (CONTINUED) 

Total Test Atmosphere Generation Period: 314 min 

Animal Exposure Time: 240 min 

Test Item Air Flow Rate: 0.086 L/min 

O2 Air Flow Rate: N/A 

Dilution Air Flow Rate: 5.43 L/min 

DOSE 

GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

SAMPLE TIMEA

 

(MIN) SAMPLE CODE

EXPOSURE CHAMBER 

CONCENTRATIONB

(PPM) 

MEAN CHAMBER 

CONCENTRATIONC

(PPM) 

0 Group 1 Sample 1 24239 

3 Group 1 Sample 2 23983 

15 Group 1 Sample 3 23273 

17 Group 1 Sample 4 23292 

28 Group 1 Sample 5 23307 

30 Group 1 Sample 6 22849 

43 Group 1 Sample 7 25055 

46 Group 1 Sample 8 26670 

56 Group 1 Sample 9 26642 

59 Group 1 Sample 10 26427 

73 Group 1 Sample 11 25734 

78 Group 1 Sample 12 26084 

85 Group 1 Sample 13 26098 

88 Group 1 Sample 14 26455 

102 Group 1 Sample 15 26083 

105 Group 1 Sample 16 25950 

116 Group 1 Sample 17 25940 

118 Group 1 Sample 18 25814 

1 

(24,000)

133 Group 1 Sample 19 25357 

25,406 

(963)

One male rat and one female rat were exposed half an hour earlier than the rest of the animals in the group to allow clinical 

observation for potentially adverse effect of test item. 

A

 = time relative to commencement of animal exposure. 

B

 = exposure chamber atmosphere of test item was sampled and measured using a gas chromatograph (GC) connected to the 

intake of the dosing chamber. 

C

 = standard deviation values presented in parentheses. 
Test Facility Study No. 

33 

TABLE 2 E XPOSURE CHAMBER CONCENTRATIONS (CONTINUED) 

Total Test Atmosphere Generation Period: 314 min 

Animal Exposure Time: 240 min 

Test Item Air Flow Rate: 0.086 L/min 

O2 Air Flow Rate: N/A 

Dilution Air Flow Rate: 5.43 L/min 

DOSE 

GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

SAMPLE TIMEA

 

(MIN) SAMPLE CODE

EXPOSURE CHAMBER 

CONCENTRATIONB

(PPM) 

MEAN CHAMBER 

CONCENTRATIONC

(PPM) 

135 Group 1 Sample 20 25882 

146 Group 1 Sample 21 26583 

150 Group 1 Sample 22 26005 

164 Group 1 Sample 23 25495 

167 Group 1 Sample 24 25810 

182 Group 1 Sample 25 25470 

185 Group 1 Sample 26 25642 

198 Group 1 Sample 27 25480 

201 Group 1 Sample 28 25335 

211 Group 1 Sample 29 25453 

215 Group 1 Sample 30 25288 

230 Group 1 Sample 31 25281 

233 Group 1 Sample 32 25432 

244 Group 1 Sample 33 25679 

259 Group 1 Sample 34 25291 

262 Group 1 Sample 35 25537 

264 Group 1 Sample 36 25667 

1 

(24,000)

268 Group 1 Sample 37 25429 

25,406 

(963)

One male rat and one female rat were exposed half an hour earlier than the rest of the animals in the group to allow clinical 

observation for potentially adverse effect of test item. 

A

 = time relative to commencement of animal exposure. 

B

 = exposure chamber atmosphere of test item was sampled and measured using a gas chromatograph (GC) connected to the 

intake of the dosing chamber. 

C

 = standard deviation values presented in parentheses. 
Test Facility Study No. 

34 

TABLE 2 E XPOSURE CHAMBER CONCENTRATIONS (CONTINUED) 

Total Test Atmosphere Generation Period: 292 min 

Animal Exposure Time: 240 min 

Test Item Air Flow Rate: 0.076 L/min 

O2 Air Flow Rate: N/A 

Dilution Air Flow Rate: 4.79 L/min 

DOSE 

GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

SAMPLE TIMEA

 

(MIN) SAMPLE CODE

EXPOSURE CHAMBER 

CONCENTRATIONB

(PPM) 

MEAN CHAMBER 

CONCENTRATIONC

(PPM) 

3 Group 4 Sample 1 15470 

6 Group 4 Sample 2 14986 

16 Group 4 Sample 3 14627 

19 Group 4 Sample 4 14930 

32 Group 4 Sample 5 14437 

35 Group 4 Sample 6 14351 

46 Group 4 Sample 7 14403 

50 Group 4 Sample 8 13875 

67 Group 4 Sample 9 15054 

69 Group 4 Sample 10 14869 

82 Group 4 Sample 11 15274 

86 Group 4 Sample 12 14762 

105 Group 4 Sample 13 15739 

109 Group 4 Sample 14 14387 

4 

(15,000)

118 Group 4 Sample 15 14453 

14,642 

(391)

A

 = time relative to commencement of animal exposure. 

B

 = exposure chamber atmosphere of test item was sampled and measured using a gas chromatograph (GC) connected to the 

intake of the dosing chamber. 

C

 = standard deviation values presented in parentheses. 
Test Facility Study No. 

35 

TABLE 2 E XPOSURE CHAMBER CONCENTRATIONS (CONTINUED) 

Total Test Atmosphere Generation Period: 292 min 

Animal Exposure Time: 240 min 

Test Item Air Flow Rate: 0.076 L/min 

O2 Air Flow Rate: N/A 

Dilution Air Flow Rate: 4.79 L/min 

DOSE 

GROUP/TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

SAMPLE TIMEA

 

(MIN) SAMPLE CODE

EXPOSURE CHAMBER 

CONCENTRATIONB

(PPM) 

MEAN CHAMBER 

CONCENTRATIONC

(PPM) 

121 Group 4 Sample 16 14462 

138 Group 4 Sample 17 14922 

141 Group 4 Sample 18 14579 

146 Group 4 Sample 19 14162 

149 Group 4 Sample 20 14449 

163 Group 4 Sample 21 14417 

171 Group 4 Sample 22 14290 

180 Group 4 Sample 23 14608 

189 Group 4 Sample 24 14603 

208 Group 4 Sample 25 14697 

212 Group 4 Sample 26 14606 

224 Group 4 Sample 27 14702 

228 Group 4 Sample 28 14259 

237 Group 4 Sample 29 14591 

4 

(15,000)

240 Group 4 Sample 30 14305 

14,642 

(391)

A

 = time relative to commencement of animal exposure. 

B

 = exposure chamber atmosphere of test item was sampled and measured using a gas chromatograph (GC) connected to the 

intake of the dosing chamber. 

C

 = standard deviation values presented in parentheses. 
Test Facility Study No. 

36 

TABLE 3 N OMINAL ATMOSPHERE CONCENTRATIONS

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

TOTAL 

GENERATION 

TIME 

(MIN) 

TOTAL AIR 

FLOW 

RATE 

(L/MIN) 

WEIGHT OF 

TEST ITEM 

USED 

(GRAM) 

CONCENTRATION 

(MG/M3) 

NOMINAL 

CONCENTRATION 

(PPM) 

MEAN CHAMBER 

CONCENTRATION 

(PPM) 

3 

(125,000) 

151 4.433-

5.429 622.5 855658 127,783 121,979 

2 

(50,000) 

31 5. 003 63 406208 60,663 49,003 

1 

(24,000) 

314 5. 516 418 241336 36,041 25,406 

4 

(15,000) 

292 4. 996 243 166572 24,876 14,642 

Note: the total generation time, total air flow rate and weight of test item used and nominal concentration for target atmosphere 

concentration of 1 25,000ppm c over both t est atmosphere optimisation period and animal i nhalation d osing p eriod. S ome 

values of total air flow rate during optimisation are estimates based on presumed values. 

Test item concentration (ppm) was calculated based on the formula: ppm = (concentration at mg/m3

 x 24.5)/MW of 
Test Facility Study No. 

37 

TABLE 4 M ORTALITY

MORTALITY DATA

DOSE GROUP/TARGET ATMOSPHERE CONCENTRATION 

(PPM) MALES FEMALES TOTAL

3 

(125,000) 5 5 10 

2 

(50,000) 2 0 2 

1 

(24,000) 1 0 1 

4 

(15,000) 0 0 0 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS

NUMBER OF ANIMALS AFFECTED/TIME

DURING EXPOSURE

PREDOSE 0 MIN 20 MIN

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F 

NAD 5 5 

Fast Respiration 5 5 

Extreme Agitation 5 5 

Tail Flicking 5 5 

Very Slow Respiration 5 5 

Very Pale Appearance 5 5 

Red/Brown Staining 

Around Eyes 5 5 

Slight Red Staining 

Around Mouth 2 3 

Marked Red Staining 

Around Mouth 2 1 

Unkempt Coat 5 5 

3 

(125,000) 

FD 5 4 

NAD = no abnormalities detected FD = found dead M = male F = female Note: inhalation exposure terminated at 20 min into dosing due to high mortality 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS 

AFFECTED/TIME

DAY 0

IMMEDIATELY 

POST DOSE

POST EXPOSURE

(CA 0.5H)

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM)

OBSERVATION

M F M F 

Subdued 1 1 

Laboured Respiration 1 1 

Piloerected 1 1 

Red Liquid from Mouth 1 1 

Red Liquid from Eyes 1 1 

Cracking Respiration 1 

Eyes bulging 1 

Vocalising 1 

Convulsing 1 

3 

(125,000) 

KP 1 

KP = killed prematurely M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DURING EXPOSURE

PREDOSE 0 MIN 1- 4 MIN

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F 

NAD 5 5 

Extreme Agitation 5 5 5 5 

Tail Flicking 5 5 5 5 

Red Liquid from 

Mouth 5 5 5 5 

Gasping 5 5 

Convulsions 5 5 

Pale 1 

Prostrate 1 

Moribund 1 

2 

(50,000) 

FD 1 

NAD = no abnormalities detected FD = found dead M = male F = female Note: inhalation exposure terminated at 4 min into dosing due to clinical signs and mortality 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 0 

IMMEDIATELY 

POST DOSE

POST EXPOSURE

(CA 1H) 

POST EXPOSURE

(CA 2H) 

POST EXPOSUR

(CA 3H) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F 

NAD 5 

Prostrate 3 1 

Pale 3 3 

Eyes Dull 3 2 

Laboured Breathing 3 4 2 2 

Red Liquid from Mouth 3 2 

Salivation 3 2 3 

Subdued 3 3 5 3 

Red Staining around Mouth 3 5 3 3 

Red Extremities 4 3 3 

Gasping 1 

Red Staining around Eyes 2 3 5 3 3 1 

Irregular Breathing 1 5 1 

Hunched 5 2 3 

2 

(50,000) 

FD 1 

NAD = no abnormalities detected; M = male; F = female Note: no observation was undertaken, in error, for females at ca 2h post exposure.
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 1 D AY 2 D AY 3 D AY 4 D AY 5 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M 

2 

(50,000) NAD 3 5 3 5 3 5 3 5 3 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 8 D AY 9 D AY 10 D AY 11 D AY 12 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M 

2 

(50,000) NAD 3 5 3 5 3 5 3 5 3 5 3 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED)

NUMBER OF ANIMALS AFFECTED/TIME

DURING EXPOSURE

PREDOSE

0 MIN 2 MIN 5- 6 MINS 9- 10 MINS 16 MINS CA 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M F M F M 

NAD 5 5 4 3 2 2 3 4 3 

Tail Flicking 1 2 3 3 1 1 1 1 3 

Struggling in Tube 1 1 1 1 1 

Laboured Breathing 1 1 1 3 4 

Pale 1 1 2 2 

Salivation 1 1 3 1 1 5 4 

Gasping 1 

Eyes Dark 1 4 

Red Liquid from 

Mouth 4 

Irregular Breathing 

1 

(24,000) 

FD 

NAD = no abnormalities detected FD = found dead M = male; F = female Note: no observation was undertaken for 4 males and 4 females at 16 min into dosing 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DURING EXPOSURE

CA 90 MIN CA 120 MIN 150 MIN CA 180 MIN CA 210MIN CA 240MIN

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M F M F 

NAD 1 1 1 1 1 1 

Red Liquid from 

Mouth 3 5 3 5 3 5 2 2 3 5 3 5 

Salivation 1 1 1 3 5 3 5 3 5 

Eyes Dark 3 4 3 4 3 4 3 4 3 4 3 4 

Irregular Breathing 3 4 3 4 

Shallow Breathing 3 4 3 5 4 5 3 5 

1 

(24,000) 

Relaxed 3 4 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 0 

IMMEDIATELY 

POST DOSE

POST EXPOSURE

(CA 15 MIN) 

POST EXPOSURE

(CA 30 MIN) 

POST EXPOSURE

(CA 2H) 

P

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F 

NAD 

Hunched 4 5 4 5 4 5 4 5 4 5 

Piloerection 2 2 3 5 3 5 2 1 2 2 

Irregular Breathing 3 2 2 3 3 5 2 2 2 4 

Staining Around 

Nose/Eyes/Muzzle 1 1 1 1 1 1 1 

Red Staining Around Head and 

Thorax 1 1 1 1 

Salivation 3 5 

Unkempt Coat 4 5 3 5 2 4 2 4 

Rolling Gait 3 5 4 5 4 5 2 4 2 4 

Subdued 2 1 2 3 3 4 2 5 2 4 

Eyes Partially Closed 1 2 4 3 3 2 4 2 4 

Fur Staining 1 2 3 2 3 2 3 2 3 

Walking on Tip Toes 2 1 

1 

(24,000) 

Tremors in Cage 

NAD = no abnormalities detected; M = male; F = female Note: the clinical observations of 1-2 h post exposure were not reported due to overlapping of timepoints. 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 1 D AY 2 D AY 3 D AY 4 D AY 5 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M 

1 NAD 4 3 4 4 4 4 4 5 4 5 4 

(24,000) Unkempt Coat 2 1 1 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 8 D AY 9 D AY 10 D AY 11 D AY 12 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M 

1 

(24,000) NAD 4 5 4 5 4 5 4 5 4 5 4 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DURING EXPOSURE

PREDOSE

0 MIN 30 MIN 60 MIN 90 MIN 120 MIN 150 M

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M F M F M F 

NAD 5 5 3 2 5 5 5 5 5 5 5 5 5 5 5 5 

4 

(15,000) Tail Flicking 2 3 

NAD = no abnormalities detected M = male F = female Note: all animals NAD at 6 min into inhalation dosing. 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 0 

IMMEDIATELY POST 

DOSE

POST EXPOSURE

(CA 0.5H) 

POST EXPOSURE

(CA 3H) 

P

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M 

4 

(15,000) NAD 5 5 5 5 5 5 5 5 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 1 D AY 2 D AY 3 D AY 4 D AY 5 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M 

4 

(15,000) NAD 5 5 5 5 5 5 5 5 5 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

TABLE 5 C LINICAL OBSERVATIONS (CONTINUED) 

NUMBER OF ANIMALS AFFECTED/TIME

DAY 8 D AY 9 D AY 10 D AY 11 D AY 12 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

OBSERVATION

M F M F M F M F M 

NAD 5 4 5 4 5 4 5 4 5 4 5 

4 

(15,000) Lump on Ventral Abdomen (10x10mm) 1 1 1 1 

NAD = no abnormalities detected M = male F = female 
Test Facility Study No. 

53 

TABLE 6 B ODY WEIGHTS (G) 

PRE 

DOSE 

(DAY) 

OBSERVATION PERIOD 

(DAY) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

ANIMAL 

NO./SEX

0 1 3 7 10 14 

Gain 

(Day 0-14) 

21M 307 N/A N/A N /A N /A N /A N/A 

22M 266 N/A N/A N /A N /A N /A N/A 

23M 276 N/A N/A N /A N /A N /A N/A 

24M 253 N/A N/A N /A N /A N /A N/A 

25M 256 N/A N/A N /A N /A N /A N/A 

Mean 272 N/A N/A N /A N /A N /A N/A 

SD 22 N/A N/A N/ A N/ A N/ A N/A 

26F 186 N/A N/A N /A N /A N /A N/A 

27F 181 N/A N/A N /A N /A N /A N/A 

28F 184 N/A N/A N /A N /A N /A N/A 

29F 181 N/A N/A N /A N /A N /A N/A 

30F 194 N/A N/A N /A N /A N /A N/A 

Mean 185 N/A N/A N /A N /A N /A N/A 

3 

(125,000) 

SD 5 N/A N/A N/ A N/ A N/ A N/A 

M = male 

F = female 

SD = standard deviation 

N/A = not applicable due to premature death 
Test Facility Study No. 

54 

TABLE 6 B ODY WEIGHTS (G) (CONTINUED) 

PRE 

DOSE 

(DAY) 

OBSERVATION PERIOD 

(DAY) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

ANIMAL 

NO./SEX

0 1 3 7 10 14 

Gain 

(Day 0-14) 

11M 293 N/A N/A N /A N /A N /A N/A 

12M 343 N/A N/A N /A N /A N /A N/A 

13M 320 322 347 366 385 395 75 

14M 293 275 277 303 336 363 70 

15M 273 270 285 308 319 337 64 

Mean 304 289 303 326 347 365 70 

SD 27 29 38 35 34 29 6 

16F 194 194 203 207 213 216 22 

17F 193 201 217 236 258 253 60 

18F 219 220 228 234 234 235 16 

19F 206 207 217 220 243 225 19 

20F 196 197 204 204 214 218 22 

Mean 202 204 214 220 232 229 28 

2 

(50,000) 

SD 11 10 10 15 19 15 18 

M = male 

F = female 

SD = standard deviation 

N/A = not applicable due to premature death 
Test Facility Study No. 

55 

TABLE 6 B ODY WEIGHTS (G) (CONTINUED) 

PRE 

DOSE 

(DAY) 

OBSERVATION PERIOD 

(DAY) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

ANIMAL 

NO./SEX

0 1 3 7 10 14 

Gain 

(Day 0-14) 

1M 228 232 247 273 296 316 88 

2M 246 230 254 280 301 330 84 

3M 259 245 266 296 325 354 95 

4M 242 N/A N/A N /A N /A N /A N/A 

5M 245 243 268 299 318 346 101 

Mean 244 238 259 287 310 337 92 

SD 11 8 10 13 14 17 8 

6F 185 181 194 214 229 242 57 

7F 189 162 194 223 246 246 57 

8F 173 167 185 203 228 233 60 

9F 170 164 182 204 226 242 72 

10F 180 177 185 206 198 217 37 

Mean 179 170 188 210 225 236 57 

1 

(24,000) 

SD 8 8 6 8 17 12 13 

M = male 

F = female 

SD = standard deviation 

N/A = not applicable due to premature death 
Test Facility Study No. 

56 

TABLE 6 B ODY WEIGHTS (G) (CONTINUED) 

PRE 

DOSE 

(DAY) 

OBSERVATION PERIOD 

(DAY) 

DOSE GROUP/

TARGET 

ATMOSPHERE 

CONCENTRATION 

(PPM) 

ANIMAL 

NO./SEX

0 1 3 7 10 14 

Gain 

(Day 0-14) 

31M 251 256 277 318 338 371 120 

32M 231 233 256 283 299 324 93 

33M 246 255 271 310 332 361 115 

34M 238 235 256 284 316 346 108 

35M 255 243 263 298 318 330 75 

Mean 244 244 265 299 321 346 102 

SD 10 11 9 16 15 20 18 

36F 216 213 219 233 241 253 37 

37F 217 215 226 238 247 265 48 

38F 186 189 194 211 214 228 42 

39F 195 193 198 211 241 228 33 

40F 202 198 207 224 229 243 41 

Mean 203 202 209 223 234 243 40 

4 

(15,000) 

SD 13 12 14 12 13 16 6 

M = male 

F = female 

SD = standard deviation 
Test Facility Study No 

TABLE 7 N ECROPSY FINDINGS

GROUP TOTALS 

 M ales F

NECROPSY FINDINGS GROUP Grp 1 Grp 2 Grp 3 Grp 4 Grp 1 Grp 

DOSE 24000 50000 125000 15000 24000 50000 

ppm pp m ppm pp m ppm pp m 

GENERAL COMMENTS 

Number of animals necropsied 5 5 5 5 5 5 

CRANIUM 

Contains blood 1 

INTESTINES 

Prominent 1 

LIVER 

Dark 1 1 1 Pale focus Enlarged 1 Prominent lobulation 1 

LUNG 

Spongy 2 

The absence of a numeral indicates that the lesion specified was not identified 
Test Facility Study No 

TABLE 7 N ECROPSY FINDINGS (CONTINUED)

GROUP TOTALS 

 M ales F

NECROPSY FINDINGS GROUP Grp 1 Grp 2 Grp 3 Grp 4 Grp 1 Grp 

DOSE 24000 50000 125000 15000 24000 50000 

ppm pp m ppm pp m ppm pp m 

LUNG 

Dark 2 1 Pale focus Reddened 4 1 

LYMPH NODE (MANDIBULAR) 

Reddened 2 Enlarged 1 

PITUITARY GLAND 

Dark 4 

SKIN AND SUBCUTIS 

Reddened 1 Staining 2 3 

The absence of a numeral indicates that the lesion specified was not identified 
Test Facility Study No 

TABLE 7 N ECROPSY FINDINGS (CONTINUED)

GROUP TOTALS 

 M ales F

NECROPSY FINDINGS GROUP Grp 1 Grp 2 Grp 3 Grp 4 Grp 1 Grp 

DOSE 24000 50000 125000 15000 24000 50000 

ppm pp m ppm pp m ppm pp m 

SYSTEMIC CONDITION 

Autolysed 1 2 

TESTIS 

Dark, right 1 

THYMUS 

Speckled 1 2 5 Dark Reddened 3 

TRACHEA 

Froth filled 2 2 

URINARY BLADDER 

Distended 1 

The absence of a numeral indicates that the lesion specified was not identified 
Test Facility Study No. 

60 

FIGURE 1 S CHEMATIC DIAGRAM OF ATMOSPHERE EXPOSURE SYSTEM
Test Facility Study No. 

61 

APPENDIX 1 GL P DOCUMENTATION

UK GLP DOCUMENTATION
Test Facility Study No. 

62 

APPENDIX 2 CERTIFICATE OF ANALYSIS OF TEST ITEM

The test item was supplied by

Information pertaining to the characterization and stability of the test item along with a 

chemical retain sample are archived by 
Test Facility Study No. 

63 

APPENDIX 3 INDIVIDUAL NECROPSY FINDINGS

 P ROJECT NUMBER: 

TREATMENT: Group 1 (24,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 1 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 2 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 3 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 4 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 SYSTEM IC CONDITION : Autolysed 

 THYMUS : Speckled, right lobe 
Test Facility Study No. 

64 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 1 (24,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 5 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

65 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 1 (24,000 ppm) FEMALES 

ANIMAL NO: FINDINGS: 

 6 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 7 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 8 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 9 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

66 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 1 (24,000 ppm) FEMALES 

ANIMAL NO: FINDINGS: 

 10 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

67 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 2 (50,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 11 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LUNG : Dark, all lobes 

 SKIN AND SUBCUTIS : Staining, red, (ventral neck, chin) 

 THYMUS : Speckled, dark, (both lobes, severe) 

 T RACHEA : Froth filled 

 12 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LIVER : Dark, all lobes 

 LUNG : Dark, all lobes 

 SKIN AND SUBCUTIS : Staining, red, (ventral neck, chin) 

 THYMUS : Speckled, dark, (both lobes, severe) 

 T RACHEA : Froth filled 

 13 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

68 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 2 (50,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 14 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 15 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

69 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 2 (50,000 ppm) FEMALES 

ANIMAL NO: FINDINGS: 

 16 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 17 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 18 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 

 19 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

70 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 2 (50,000 ppm) FEMALES 

ANIMAL NO: FINDINGS: 

 20 Terminal Kill 

 Day of Necropsy: 16 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

71 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 3 (125,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 21 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LUNG : Reddened, all lobes 

 Spongy, all lobes 

 LYMPH NODE (MANDIBULAR) : Enlarged, (8 x 4 x 2 mm) 

 Reddened 

 PITUITARY GLAND : Dark 

 SKIN AND SUBCUTIS : Staining, red, (muzzle, severe) 

 TESTIS : Dark, right 

 THYMUS : Reddened, (both lobes) 

 Speckled, (both lobes) 

 TRACHEA : Froth filled, white 

 22 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 CRANIUM : Contains blood 

 LIVER : Dark, all lobes 

 LUNG : Dark, all lobes 

 PITUITARY GLAND : Dark 

 SKIN AND SUBCUTIS : Staining, red, (muzzle) 

 THYMUS : Speckled, (both lobes, slight) 
Test Facility Study No. 

72 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 3 (125,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 23 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 INTESTINES : Prominent (peyers patch) 

 LIVER : Prominent lobulation, all lobes 

 LUNG : Spongy, all lobes 

 Reddened, all lobes, (severe) 

 PITUITARY GLAND : Dark 

 SKIN AND SUBCUTIS : Reddened 

 SYSTEM IC CONDITION : Autolysed 

 THYMUS : Reddened, (both lobes) 

 Speckled, (both lobes) 

 TRACHEA : Froth filled, white 

 URINARY BLADDER : Distended, (by contents, 20 x 18 x 11 

 mm) 

 24 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LU NG : Reddened 

 SYSTEM IC CONDITION : Autolysed 

 THYM US : Speckled 

 Reddened 
Test Facility Study No. 

73 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 3 (125,000 ppm) MALES 

ANIMAL NO: FINDINGS: 

 25 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LUNG : Reddened, all lobes 

 LYMPH NODE (MANDIBULAR) : Reddened 

 PITUITARY GLAND : Dark 

 SKIN AND SUBCUTIS : Staining, red, (muzzle, head) 

 THYMUS : Speckled, dark, (both lobes) 
Test Facility Study No. 

74 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 3 (125,000 ppm) FEMALES 

ANIMAL NO: FINDINGS: 

 26 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LU NG : Reddened 

 LYMPH NODE (MANDIBULAR) : Reddened 

 PITUITARY GLAND : Dark 

 THYM US : Speckled 

 Reddened 

 27 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LUNG : Reddened, all lobes 

 SKIN AND SUBCUTIS : Staining, red, (muzzle) 

 THYMUS : Speckled, dark, (both lobes) 

 2 8 Killed Prematurely 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LIVER : Dark, all lobes 

 LUNG : Reddened, all lobes 

 SKIN AND SUBCUTIS : Staining, red, (muzzle) 

 THYMUS : Speckled, left lobe, dark 
Test Facility Study No. 

75 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 3 (125,000 ppm) FEMALES 

ANIMAL NO: FINDINGS: 

 29 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 INTESTINES : Prominent (peyers patch) 

 LIVER : Pale focus, caudate lobe, white, (one, 

 3 mm) 

 LUNG : Reddened, all lobes 

 Pale focus, right middle lobe, (two, 

 2 mm) 

 LYMPH NODE (MANDIBULAR) : Reddened 

 PITUITARY GLAND : Dark 

 THYMUS : Dark, (both lobes) 

 30 Found Dead 

 Day of Necropsy: 1 

 NECRO PSY FINDINGS: 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LIVER : Dark, all lobes 

 LUNG : Reddened, all lobes 
Test Facility Study No. 

76 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 4 (15,000 ppm) MALES 

ANIMAL NO: NECROPSY FINDINGS: 

 31 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 

 32 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 

 33 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LIVER : Enlarged, all lobes, (75 x 60 x 19 

 mm) 

 Dark, all lobes 

 LUNG : Reddened, all lobes 

 34 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

77 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 4 (15,000 ppm) MALES 

ANIMAL NO: NECROPSY FINDINGS: 

 35 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

78 

APPENDIX 3 I NDIVIDUAL NECROPSY FINDINGS

(CONTINUED) 

 P ROJECT NUMBER: 

TREATMENT: Group 4 (15,000 ppm) FEMALES 

ANIMAL NO: NECROPSY FINDINGS: 

 36 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : Tissues not listed below were normal 

 LUNG : Dark, all lobes 

 37 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 

 38 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 

 39 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 

 40 Terminal Kill 

 Day of Necropsy: 16 

 GENERAL COMMENTS : All tissues normal 
Test Facility Study No. 

79 

APPENDIX 4 DEVELOPMENT AND VALIDATION OF METHODOLOGIES FOR THE 

ANALYSIS OF IN INHALATION TOXICOLOGY STUDIES 
Test Facility Study No. 

80 
Test Facility Study No. 

81 
Test Facility Study No. 

82 
Test Facility Study No. 

83 
Test Facility Study No. 

84 
Test Facility Study No. 

85 
Test Facility Study No. 

86 
Test Facility Study No. 

87 
Test Facility Study No. 

88 
Test Facility Study No. 

89 
Test Facility Study No. 

90 
Test Facility Study No. 

91 
Test Facility Study No. 

92 
Test Facility Study No. 

93 
Test Facility Study No. 

94 
Test Facility Study No. 

95 
Test Facility Study No. 

96 
Test Facility Study No. 

97 
Test Facility Study No. 

98 
Test Facility Study No. 

99 
Test Facility Study No. 

100 
Test Facility Study No. 

101 
Test Facility Study No. 

102 
Test Facility Study No. 

103 
Test Facility Study No. 

104 
Test Facility Study No. 

105 
Test Facility Study No. 

106 
Test Facility Study No. 

107 
Test Facility Study No. 

108 
Test Facility Study No. 

109 
Test Facility Study No. 

110 
Test Facility Study No. 

111 
Test Facility Study No. 

112 
Test Facility Study No. 

113