## Test Facility Study No.

Page 1 of 113 Study Title



# A CUTE 

# I NHALATION 

# T OXICITY 

# S TUDY IN 

# R ATS



## 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 

# S TATEMENTS OF 

# C ONFIDENTIALITY



## 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:



# S TATEMENT OF 

# N O 

# D ATA 

# C ONFIDENTIALITY



## 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 

# T

ABLE OF 

# C ONTENTS



## 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 C 50 

## Estimation .................................................................................... 24

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

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



# T ABLES



## 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



# F IGURES



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



# A PPENDICES



## 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

An alysis of in Inhalation Toxi cology Studies................ 79

## Test Facility Study No.

8 

# L IST OF 

# G ENERAL 

# A BBREVIATIONS AND 

# S YMBOLS



## g gr am(s)

GLP Good Laboratory Practice

h h our(s)

L l itre(s)

m 3 

## 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 

# B ASIC 

# S TUDY 

# I NFORMATION



## 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

S TUDY 

### D IRECTOR 

### T EST 

### F ACILITY 

### A DDRESS



## Sponsor Monitor

S PONSOR 

### M ONITOR 

### C OMPANY 

### N AME 

### A DDRESS

## Test Facility Study No.

11 

# A CUTE 

# I NHALATION 

# T OXICITY 

# S TUDY IN 

# R ATS



# 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 LC 50 

## 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 

# M ETHODS



## 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.

Te st 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 15.90-27.19 o 

## 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:

A NIMAL 

### N UMBERS


### D OSE 

### G ROUP 

### T ARGET 

### A TMOSPHERE 

### C ONCENTRATION 

### ( PPM 

### ) M ALES 

### F EMALES



### 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

fr om stainless steel. This design allowed continuous supply of test atmosphere tobe 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 (m 3 

## ) 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 

# F ROM 

# P ROTOCOL



## 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.

Gro up mean values of exposure chamber temperature, relative humidity and oxygen levels are as follows:

D OSE 

### G ROUP 

### /T ARGET


### A

TMOSPHERE 

### C ONCENTRATION 

### ( PPM 

### ) T EMPERATURE A


### ( ° C) R ELATIVE 

### H UMIDITY A


### (%) O2 A


### (%)

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 D OSE 

### G ROUP 

### /T ARGET


### A

TMOSPHERE 

### C ONCENTRATION 

### ( PPM 

### ) A CHIEVED 

### A TMOSPHERE 

### C ONCENTRATION A


### ( PPM 

### ) N OMINAL 

### C ONCENTRATION 

### ( 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 8 th 

## 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

pres ented 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 C 50 

## Estimation
Based on the findings, the LC 50 

## 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 LC 50 

## was estimated to be between 25,400 ppm and 49,000 ppm.

## Test Facility Study No.

25 

# 7.0 R ETENTION OF 

# R ECORDS



## 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 

# T

ABLE 

# 1 E XPOSURE 

# C HAMBER 

# A IR 

# F LOW 

# R ATE


# , T EMPERATURE


# , H UMIDITY AND 

# O XYGEN 

# L EVEL

D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) T ARGET T IME D URING E XPOSURE A ( MIN ) A IR FLOW ( L / MIN ) T EMPERATURE ( O C) R ELATIVE H UMIDITY (%) O 2 (%)

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 

# T

ABLE 

# 1 E XPOSURE 

# C HAMBER 

# A IR 

# F LOW 

# R ATE


# , T EMPERATURE


# , H UMIDITY AND 

# O XYGEN 

# L EVEL 

# ( CONTINUED 

# )

D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) T ARGET T IME D URING E XPOSURE A ( MIN ) A IR FLOW ( L / MIN ) T EMPERATURE ( O C) R ELATIVE H UMIDITY (%) O 2 (%)

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 

# T

ABLE 

# 1 E XPOSURE 

# C HAMBER 

# A IR 

# F LOW 

# R ATE


# , T EMPERATURE


# , H UMIDITY AND 

# O XYGEN 

# L EVEL 

# ( CONTINUED 

# )

D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) T ARGET T IME D URING E XPOSURE A ( MIN ) A IR FLOW ( L / MIN ) T EMPERATURE ( O C) R ELATIVE H UMIDITY (%) O 2 (%)

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 

# T

ABLE 

# 1 E XPOSURE 

# C HAMBER 

# A IR 

# F LOW 

# R ATE


# , T EMPERATURE


# , H UMIDITY AND 

# O XYGEN 

# L EVEL 

# ( CONTINUED 

# )

D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) T ARGET T IME D URING E XPOSURE A ( MIN ) A IR FLOW ( L / MIN ) T EMPERATURE ( O C) R ELATIVE H UMIDIT Y (%) O 2 (%)

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 

# T

ABLE 

# 2 E XPOSURE 

# C HAMBER 

# C ONCENTRATIONS

Total Test Atmosphere Generation Period: 72 min

Animal Exposure Time: 20 min

Test Item Air Flow Rate: 0.674 L/min

O 2 Air Flow Rate: 0.253 L/min

Dilution Air Flow Rate: 3.99 L/min

D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) S AMPLE T IME A ( MIN ) S AMPLE C ODE E XPOSURE C HAMBER C ONCENTRATION B
( PPM ) M EAN C HAMBER C ONCENTRATION C
( 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 

# T

ABLE 

# 2 E XPOSURE 

# C HAMBER 

# C ONCENTRATIONS 

# (C ONTINUED 

# )

Total Test Atmosphere Generation Period: 31 min

Animal Exposure Time: 4 min

Test Item Air Flow Rate: 0.252 L/min

O 2 Air Flow Rate: 0.073 L/min

Dilution Air Flow Rate: 4.678 L/min

D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) S AMPLE T IME A ( MIN ) S AMPLE C ODE E XPOSURE C HAMBER C ONCENTRATION B
( PPM ) M EAN C HAMBER C ONCENTRATION C
( 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 

# T

ABLE 

# 2 E XPOSURE 

# C HAMBER 

# C ONCENTRATIONS 

# (C ONTINUED 

# )

Total Test Atmosphere Generation Period: 314 min

Animal Exposure Time: 240 min

Test Item Air Flow Rate: 0.086 L/min

O 2 Air Flow Rate: N/A

Dilution Air Flow Rate: 5.43 L/min

D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) S AMPLE T IME A ( MIN ) S AMPLE C ODE E XPOSURE C HAMBER C ONCENTRATION B
( PPM ) M EAN C HAMBER C ONCENTRATION C
( 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 

# T

ABLE 

# 2 E XPOSURE 

# C HAMBER 

# C ONCENTRATIONS 

# (C ONTINUED 

# )

Total Test Atmosphere Generation Period: 314 min

Animal Exposure Time: 240 min

Test Item Air Flow Rate: 0.086 L/min

O 2 Air Flow Rate: N/A

Dilution Air Flow Rate: 5.43 L/min

D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) S AMPLE T IME A ( MIN ) S AMPLE C ODE E XPOSURE C HAMBER C ONCENTRATION B
( PPM ) M EAN C HAMBER C ONCENTRATION C
( 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 

# T

ABLE 

# 2 E XPOSURE 

# C HAMBER 

# C ONCENTRATIONS 

# (C ONTINUED 

# )

Total Test Atmosphere Generation Period: 292 min

Animal Exposure Time: 240 min

Test Item Air Flow Rate: 0.076 L/min

O 2 Air Flow Rate: N/A

Dilution Air Flow Rate: 4.79 L/min

D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) S AMPLE T IME A ( MIN ) S AMPLE C ODE E XPOSURE C HAMBER C ONCENTRATION B
( PPM ) M EAN C HAMBER C ONCENTRATION C
( 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 

# T

ABLE 

# 2 E XPOSURE 

# C HAMBER 

# C ONCENTRATIONS 

# (C ONTINUED 

# )

Total Test Atmosphere Generation Period: 292 min

Animal Exposure Time: 240 min

Test Item Air Flow Rate: 0.076 L/min

O 2 Air Flow Rate: N/A

Dilution Air Flow Rate: 4.79 L/min

D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) S AMPLE T IME A ( MIN ) S AMPLE C ODE E XPOSURE C HAMBER C ONCENTRATION B
( PPM ) M EAN C HAMBER C ONCENTRATION C
( 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 

# T

ABLE 

# 3 N OMINAL 

# A TMOSPHERE 

# C ONCENTRATIONS

D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) T OTAL G ENERATION T IME ( MIN ) T OTAL A IR F LOW R ATE (L/ MIN ) W EIGHT OF T EST I TEM

U SED ( GRAM ) C ONCENTRATION ( MG / M 3 ) N OMINAL C ONCENTRATION ( PPM ) M EAN C HAMBER C ONCENTRATION ( 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 atmosphe re 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/m 3 x 24.5)/MW of

## Test Facility Study No.

37 

# T

ABLE 

# 4 M ORTALITY

M ORTALITY D ATA D OSE G ROUP /T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) M ALES F EMALES T OTAL

3 (125,000) 5 5 10

2 (50,000) 2 0 2

1 (24,000) 1 0 1

4 (15,000) 0 0 0Test Facility Study No.38TABLE5 CLINICALOBSERVATIONSNUMBER OFANIMALS AFFECTED/TIMEDURING EXPOSUREPREDOSE0MIN20MINDOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F M FNAD5 5Fast Respiration 5 5Extreme Agitation 5 5Tail Flicking 5 5Very Slow Respiration 5 5Very Pale Appearance 5 5Red/Brown StainingAround Eyes5 5Slight Red StainingAround Mouth2 3Marked Red StainingAround Mouth2 1Unkempt Coat 5 53(125,000)FD 5 4NAD = no abnormalities detectedFD = found deadM = maleF = femaleNote: inhalation exposure terminated at 20 min into dosing due to high mortalityTest Facility Study No.39TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY0IMMEDIATELYPOSTDOSEPOSTEXPOSURE(CA0.5H)DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M FSubdued1 1Laboured Respiration 1 1Piloerected1 1Red Liquid from Mouth 1 1Red Liquid from Eyes 1 1Cracking Respiration 1Eyes bulging1Vocalising1Convulsing13(125,000)KP1KP = killed prematurelyM = maleF = femaleTest Facility Study No.40TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALS AFFECTED/TIMEDURING EXPOSUREPREDOSE0MIN1- 4MINDOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F MFNAD5 5Extreme Agitation 5 5 5 5Tail Flicking 5 5 5 5Red Liquid fromMouth5 5 5 5Gasping 5 5Convulsions 5 5Pale1Prostrate 1Moribund 12(50,000)FD 1NAD = no abnormalities detectedFD = found deadM = maleF = femaleNote: inhalation exposure terminated at 4 min into dosing due to clinical signs and mortalityTest Facility Study No.41TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY0IMMEDIATELYPOSTDOSEPOSTEXPOSURE(**CA**1H)POSTEXPOSURE(**CA**2H)POSTEXPOSURE(**CA**3H)POSTEXPOSURE(**CA**5H)DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F M F M F M FNAD5 2 5Prostrate3 1Pale3 3Eyes Dull3 2Laboured Breathing3 4 22Red Liquid from Mouth 3 2Salivation3 2 3Subdued3 3 5 3Red Staining around Mouth 3 5 33Red Extremities43 3Gasping1Red Staining around Eyes 2 3 5 33 1Irregular Breathing1 5 1Hunched5 232(50,000)FD 1NAD = no abnormalities detected; M = male; F = femaleNote: no observation was undertaken, in error, for females atca2h post exposure.Test Facility Study No.42TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F MF MF MF MF MF MF2(50,000)NAD3 5 3 5 35 3 5 35 3 5 35NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.43TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY8 DAY9 DAY10 DAY11 DAY12 DAY13 DAY14DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F MF MF MF MF MF MF2(50,000)NAD3 5 3 5 3 5 35 3 5 35 35NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.44TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDURINGEXPOSUREPREDOSE0MIN2MIN5- 6MINS9- 10MINS16MINS**CA**30MINS47- 60MINSDOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F M F M F M F M F M F M FNAD5 5 4 3 2 23 4 31Tail Flicking 1 2 3 31 1 1 1 3 2Struggling in Tube 1 1 11 1 2Laboured Breathing1 1 1 34 4Pale1 1 2 2Salivation 1 1 3 1 1 5 41Gasping 1Eyes Dark14 4 3 4Red Liquid fromMouth4 5 4 5Irregular Breathing 3 41(24,000)FD 1NAD = no abnormalities detectedFD = found deadM = male; F = femaleNote: no observation was undertaken for 4 males and 4 females at 16 min into dosingTest Facility Study No.45TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDURINGEXPOSURE**CA**90MIN**CA**120MIN150MIN**CA**180MIN**CA**210MIN**CA**240MINDOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F M F M F M F M FNAD 1 11 1 1 1Red Liquid fromMouth3 5 3 5 3 5 2 2 3 5 35Salivation 1 1 1 3 53 5 3 5Eyes Dark 3 4 3 4 3 4 3 4 3 4 34Irregular Breathing 3 4 3 4Shallow Breathing 3 4 3 5 4 5 3 51(24,000)Relaxed3 4NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.46TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY0IMMEDIATELYPOSTDOSEPOSTEXPOSURE(**CA**15MIN)POSTEXPOSURE(**CA**30MIN)POSTEXPOSURE(**CA**2H)POSTEXPOSURE(**CA**3H)POSTEXPOSURE(**CA**4H)DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F M F M FM F M FNAD 1Hunched4 5 4 5 4 54 5 4 53 4Piloerection2 2 3 5 3 52 1 2 21Irregular Breathing3 2 2 3 3 52 2 2 41Staining AroundNose/Eyes/Muzzle1 1 1 11 1 1 1 1Red Staining Around Head andThorax1 11 11 1Salivation3 5Unkempt Coat4 5 3 52 4 2 42 4Rolling Gait3 5 4 5 4 52 4 2 42 4Subdued2 1 2 3 3 42 5 2 43Eyes Partially Closed 1 2 4 3 32 4 2 43Fur Staining1 2 3 2 32 3 2 32 3Walking on Tip Toes2 1 1 1 1 11(24,000)Tremors in Cage1 1NAD = no abnormalities detected; M = male; F = femaleNote: the clinical observations of 1-2 h post exposure were not reported due to overlapping of timepoints.Test Facility Study No.47TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F MF MF MF MF MF MFNAD4 3 4 4 4 4 45 4 5 45 451(24,000)Unkempt Coat 2 1 1NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.48TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY8 DAY9 DAY10 DAY11 DAY12 DAY13 DAY14DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F MF MF MF MF MF MF1(24,000)NAD4 5 4 5 4 5 45 4 5 45 45NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.49TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDURINGEXPOSUREPREDOSE0MIN30MIN60MIN90MIN120MIN150MIN180MIN210MIN240MINDOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F M F M F M F M F M F M F M F M FNAD 5 5 3 2 5 5 5 55 5 5 5 5 5 5 55 5 5 54(15,000)Tail Flicking 2 3NAD = no abnormalities detectedM = maleF = femaleNote: all animals NAD at 6 min into inhalation dosing.Test Facility Study No.50TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY0IMMEDIATELYPOSTDOSEPOSTEXPOSURE(CA0.5H)POSTEXPOSURE(**CA**3H)POSTEXPOSURE(**CA**7H)DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F M F MF MF4(15,000)NAD5 5 5 5 5 5 5 5NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.51TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F MF MF MF MF MF MF4(15,000)NAD5 5 5 5 55 5 5 55 5 5 55NAD = no abnormalities detectedM = maleF = femaleTest Facility Study No.52TABLE5 CLINICALOBSERVATIONS(CONTINUED)NUMBER OFANIMALSAFFECTED/TIMEDAY8 DAY9 DAY10 DAY11 DAY12 DAY13 DAY14DOSEGROUP/TARGETATMOSPHERECONCENTRATION(PPM)OBSERVATIONM F MF MF MF MF MF MFNAD5 4 5 4 5 4 54 5 4 54 544(15,000)Lump on VentralAbdomen (10x10mm)1111111NAD = no abnormalities detectedM = maleF = female

## Test Facility Study No.

53 

# T

ABLE 

# 6 B ODY 

# W EIGHTS 

# ( G 

# )

P RE D OSE (D AY ) O BSERVATION P ERIOD (D AY ) D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) A NIMAL N O ./S EX

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 

# T

ABLE 

# 6 B ODY 

# W EIGHTS 

# ( G 

# ) ( CONTINUED 

# )

P RE D OSE (D AY ) O BSERVATION P ERIOD (D AY ) D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) A NIMAL N O ./S EX

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 

# T

ABLE 

# 6 B ODY 

# W EIGHTS 

# ( G 

# ) ( CONTINUED 

# )

P RE D OSE (D AY ) O BSERVATION P ERIOD (D AY ) D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) A NIMAL N O ./S EX

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 

# T

ABLE 

# 6 B ODY 

# W EIGHTS 

# ( G 

# ) ( CONTINUED 

# )

P RE D OSE (D AY ) O BSERVATION P ERIOD (D AY ) D OSE G ROUP /

T ARGET A TMOSPHERE C ONCENTRATION ( PPM ) A NIMAL N O ./S EX

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 deviationTest Facility Study No57TABLE7 NECROPSYFINDINGSGROUP TOTALSMalesFemalesNECROPSY FINDINGS GROUP Grp 1 Grp 2 Grp 3 Grp 4 Grp 1 Grp 2 Grp 3 Grp 4DOSE24000 50000 125000 15000 24000 50000 125000 15000ppm pp m ppm pp m ppm pp m ppm pp mGENERAL COMMENTSNumber of animals necropsied5 55 55 55 5CRANIUMContains blood1INTESTINESProminent11LIVERDark11 12Pale focus1Enlarged1Prominent lobulation1LUNGSpongy2The absence of a numeral indicates that the lesion specified was not identifiedTest Facility Study No58TABLE7 NECROPSYFINDINGS(CONTINUED)GROUP TOTALSMalesFemalesNECROPSY FINDINGS GROUP Grp 1 Grp 2 Grp 3 Grp 4 Grp 1 Grp 2 Grp 3 Grp 4DOSE24000 50000 125000 15000 24000 50000 125000 15000ppm pp m ppm pp m ppm pp m ppm pp mLUNGDark2 11Pale focus1Reddened4 15LYMPH NODE (MANDIBULAR)Reddened22Enlarged1PITUITARY GLANDDark42SKIN AND SUBCUTISReddened1Staining2 32The absence of a numeral indicates that the lesion specified was not identifiedTest Facility Study No59TABLE7 NECROPSYFINDINGS(CONTINUED)GROUP TOTALSMalesFemalesNECROPSY FINDINGS GROUP Grp 1 Grp 2 Grp 3 Grp 4 Grp 1 Grp 2 Grp 3 Grp 4DOSE24000 50000 125000 15000 24000 50000 125000 15000ppm pp m ppm pp m ppm pp m ppm pp mSYSTEMIC CONDITIONAutolysed12TESTISDark, right1THYMUSSpeckled1 2 53Dark1Reddened31TRACHEAFroth filled2 2URINARY BLADDERDistended1The absence of a numeral indicates that the lesion specified was not identified

## Test Facility Study No.

60 

# F IGURE 

# 1 S CHEMATIC 

# D IAGRAM OF 

# A TMOSPHERE 

# E XPOSURE 

# S YSTEM

## Test Facility Study No.

61 

# A PPENDIX 

# 1 GL P D OCUMENTATION



# UK GLP D OCUMENTATION

## Test Facility Study No.

62 

# A PPENDIX 

# 2 C ERTIFICATE OF 

# A NALYSIS OF 

# T EST 

# I TEM



## 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS

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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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 

# A PPENDIX 

# 3 I NDIVIDUAL 

# N ECROPSY 

# F INDINGS 

# ( 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.

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# A PPENDIX 

# 4 D EVELOPMENT AND 

# V ALIDATION OF 

# M ETHODOLOGIES FOR THE


# A NALYSIS OF IN 

# I NHALATION 

# T OXICOLOGY 

# S TUDIES

## Test Facility Study No.

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