# FINAL REPORT

Study Title



# ASSESSMENT OF ACUTE INHALATION TOXICITY WITH

IN THE RAT

Author

Test Facility

Laboratory Pro ject Identification- Page 2 - 

### 1. CONTENTS

1. CONTENTS

...................................................................................................................................... 2

2. STATEMEN T OF GLP COMPLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE STATEMENT

............................................................................................ 4

4. SUMMARY

........................................................................................................................................ 5

CONCLUSION

.......................................................................................................................................... 6

5. INTRODUCTION

.............................................................................................................................. 7

5.1. Preface

..................................................................................................................................... 7

5.2. Aims of s tudy

............................................................................................................................ 7

5.3. Guidelines

................................................................................................................................ 7

5.4. Storage and retention of records and materials

....................................................................... 8

6. MATERIALS AND METHODS

.......................................................................................................... 9

6.1. Test substance

......................................................................................................................... 9

6.1.1. Test substance information

............................................................................................. 9

6.1.2. Study specific test substance information

...................................................................... 9

6.1.3. Test substance preparation

............................................................................................ 9

6.2. Exposure chamber

................................................................................................................... 9

6.3. Test atmosphere generation

.................................................................................................. 10

6.4. Test atmosphere characterization

.......................................................................................... 10

6.4.1. Nominal concentration

.................................................................................................. 10

6.4.2. Actual concentration

..................................................................................................... 10

6.4.3. Particle size characterizat ion

........................................................................................ 10

6.4.4. Stability monitoring

........................................................................................................ 10

6.4.5. Temperature and relative humidity

............................................................................... 10

6.5. Test system

............................................................................................................................ 11

6.6. Animal husbandry

.................................................................................................................. 11

6.7. Study design

.......................................................................................................................... 12

6.8. Treatment

............................................................................................................................... 12

6.9. Observations

.......................................................................................................................... 12

6.10. Electronic data capture

.......................................................................................................... 13

6.11. Interpretation

.......................................................................................................................... 13

6.12. List of deviations

.................................................................................................................... 13

6.12.1. List of protocol deviations

........................................................................................ 13

6.12.2. List of standard operating procedures deviations

.................................................... 13

7. RESULTS

....................................................................................................................................... 14

7.1. Test atmosphere characterization

.......................................................................................... 14

7.1.1. Concentration (APPENDIX 1, Table 1 and Figure 3)

................................................... 14

7.1.2. Particle size (APPEN DIX 1, Table 2)

............................................................................ 14

7.2. Observations

.......................................................................................................................... 14

7.2.1. Mortality (APPENDIX 1, Table 3)

.................................................................................. 14

7.2.2. Clinical Signs (APPENDIX 1, Table 4)

.......................................................................... 14

7.2.3. Body Weights (APPENDIX 1, Table 5)

......................................................................... 14

7.3. Macroscopic Findings (APPENDIX 1, Table 6)

...................................................................... 15

8. CONCLUSION

................................................................................................................................ 15

APPENDICES

APPENDIX 1 FIGURES AND TABLES- Page 5 - 

### 4. SUMMARY

Assessment of acute inhalation toxicity with in the rat

The study was carried out based on the guidelines described in:

- OECD Guidelines, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", September 2009.

- Commission Regulation (EC) No 440/2008, B.2. Acute Toxicity (inhalation), L 142, May 2008.

- EPA OPPTS 870.1300, Acute inhalation Toxicity. EPA 712 -C- 98

- 193, August 1998.

- JMAFF, 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

was administered as an aerosol by inhalation for 4 hours to two groups of five

male and five female Wistar rats each group. Animals were subjected to daily observations and

determination of body weights on Days 1, 2, 8 and 15. Macroscopic examination was performed on

the day of death or after terminal sacrifice (Day 15).

RESULTS

For the 5 mg/L exposure group, the time

- weighted mean actual concentration was 5.1 ± 0.2 mg/L. The

nominal concentration was 4.9 mg/L. The generation efficiency (ratio of actual and nominal

concentration) was 104%. Da ta obtained from the opacity monitor showed that the aerosol was

sufficiently stable.

For the 1 mg/l exposure group, the time

- weighted mean actual concentration was 1.0 ± 0.05 mg/L.

The nominal concentration was 1.15 mg/L. The generation efficiency (ratio of actual and nominal

concentration) was 87%. Data obtained from the opacity monitor showed that the aerosol was

sufficiently stable. The short drops in opacity were caused by adjustments to the generation equipment

and were considered not to have affected the exposure level.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were

determined twice. For the 5 mg/L exposure group, the MMAD was 2.9 μm (gsd 1.9) and 2.6 μm (gsd

2.1). For the 1 mg/L exposure group, the MMAD was 2.8 μm (gsd 2.1) and 2.9 μm (gsd 1.9).

At 5 mg/L, on the day following exposure (Day 2) three males and two females were found dead and

the remaining animals were sacrificed for ethical reasons

. At 1 mg/L, one female was sacrificed for ethical reasons on Day 2. No further mortality occurred.

At 5 mg/L, the animals showed gasping and laboured respiration during exposure. After exposure,

hunched posture, lethargy, flat posture, laboured respiration, piloerection, ptosis and/or moribund

status were shown b y the animals.

At 1 mg/L, no clinical signs were noted during exposure. After exposure, the animals showed lethargy,

hunched posture, piloerection and/or ptosis between Days 1 and 4. In addition, the female sacrificed

on Day 2 showed uncoordinated movements, chromodacryorrhoea, hypothermia, shallow respiration

and a lean appearance.

At 5 mg/L, body weights loss was noted in the animals found dead or sacrificed on Day 2. At 1 mg/L,

o verall body weight gain and body weight loss in males and females was within the range expected for

rats of this strain and age used in this type of study.

At 5 mg/L, macroscopic post mortem examination of the animals that were found dead or sacrificed for

ethical reasons during the study revealed abnormalities of the lungs (many dark red foci), stomach

(black foci in the glandular mucosa), liver (pale discoloration) and mandibular lymph nodes (dark red

discoloration). At 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the

animals.- Page 6 - 

### CONCLUSION

The inhalatory LC 50, 4h 

### value of in Wistar rats was established to be within the

range of 1

– 5 mg/L.

Based on these results and according to the:

- Globally harmonized system of classification of chemicals (GHS), United Nations, New York and

Geneva (2011)

, should be classified in Class 4 for acute toxicity by the

inhalation route.

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and

mixtures, should be classified as Category 4 and should be labeled as H332:

Harmful if inhaled.- Page 7 - 

### 5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

St udy Plan (in

- life phase) Start:

Completion: 21 November 2011

07 December 2011

5.2. Aims of study

The objective of this study was to assess the toxicity of the test substance in rats following inhalation

exposure to one or more defined concentrations for a single period of 4 hours. The results of the study

allow the test substance to be ranked according to most classification systems currently in use. This

study should provide a rational basis for risk assessment in man. The inhalatory route was selected,

as it is a possible route of human exposure during manufacture, handling or use of the test substance.

5.3. Guidelines

The protocol was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical

Committee of as required by the Dutch Act on Animal Experimentation

(February 1997). The study was conducted based on the following guidelines:

Organisation for Economic Co

- operation and Development (OECD), OECD Guidelines for Testing of

Chemicals, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", September 2009.

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other

Health Effects ; B.2. AcuteToxicity (inhalation). Official Journal of the European Communities No. L142,

Ma y 2008, including most recent amendments.

EPA Health Effects Test Guidelines OPPTS 870.1300, Acute inhalation Toxicity. EPA 712 -C- 98

- 193,

August 1998.

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147,

Apr il 2011, including the most recent partial revisions.- Page 8 - 

### 5.4. Storage and retention of records and materials

Records and materials pertaining to the study including protocol, raw data and the final report are

retained in the archives for a period of at least 2 years after finalization of the report. After this

period, the sponsor will be contacted to determine how the records and materials should be handled.

will retain information concerning decisions made.

will retain a test substance sample until the expiry date, but no longer than 10 years after

finalization of the report. After this period the sample will be destroyed.- Page 9 - 

### 6. MATERIALS AND METHODS

6.1. Test substance

6.1.1. Test substance information

Identification

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012

6.1.2. Study specific test substance information

Hygroscopic No

Volatile No

pH (1% in water, indicative value ) (determined at

Test substance handling Flush contain er with nitrogen after handling

Specific Gravity / Density

Stability at higher temperatures Yes, maximum temperature: 130 ° C

maximum duration: 24 hours under nitrogen seal

6.1.3. Test substance preparation

The test substance was used as delivered by the sponsor.

6.2. Exposure chamber

The design of the exposure chamber is based on the flow past nose

- only inhalation chamber (Am. Ind.

Hyg Assoc. J. 44(12): 923

- 928, 1983). The chamber consisted of three animal sections with eight

animal ports each ( APPENDIX 1

, Figure 1 ) . Each animal port had its own atmosphere inlet and

exhaust outlet. The animals were placed in restraining tubes and connected to the animal ports. The

number of animal sections and number of open inlets were adapted to the air flow in such a way that

at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen

supply to the animals. The main inlet of the test atmosphere was located at the top section and the

main outlet was located at the bottom section. The direction of the flow of the test atmosphere

guaranteed a freshly generated atmosphere for each individual animal.

The placement of the individual animals in the inhalation chamber is shown in Appendix 1 (Figure 2).

All components of the exposure chamber in contact with the test material were made of stainless steel,

glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the

exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.- Page 10

- 

### 6.3. Test atmosphere generation

The test substance was transferred to a nebulizer (LC SPRINT Baby rood, Pari, Starnberg, Germany)

by means of a rotating pump (type VL500 digit, VERDER Lab Tec GmbH & Co. KG, Haan, Germany)

and nebulized with pressurized air. The primary aerosol was diluted with humidified pressurized air

and passed through the exposure chamber. The mean total airflow was 19 L/min for the 5 mg/L

exposure group and 34 L/min for the 1 mg/L exposure group. (Appendix 1, Figure 1).

From the exposure chamber the test atmosphere was passed through a filter before it was released to

the exhaust of the fume hood.

6.4. Test atmosphere characterization

6.4.1. Nominal concentration

The nominal concentration was calculated by dividing the amount of test substance used by the

volume of pressurized air (average air flow times exposure time) entering the exposure chamber used

for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time

was negligible. The volume of air was calculated from the average air flow (measured by means of

thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the

exposure time.

6.4.2. Actual concentration

A to tal of 16 and 11 samples were taken for determination of the actual concentration during exposure

at 5 and 1 mg/L, respectively. Samples were drawn from the test atmosphere through a tube mounted

in one of the free animal ports of the middle section of the exposure chamber. Samples were drawn

through a glass fiber filter (type APFC04700, Millipore, Billerica, MA, USA). The collected amount test

substance in the air sample was measured gravimetrically. Sample volumes were measured by means

of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands).

Subsequently the time

- weighted mean concentrations with the standard deviations were calculated.

6.4.3. Particle size characterization

The particle size distribution was characterized twice during each exposure period. The samples were

drawn at 2 L/min from the test atmosphere through a tube mounted in one of the free animal ports of

the middle section of the exposure chamber (Appendix 1, Figures 1 and 2). The samples were

collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (SKC 225

- 713, fiber glass, SKC Omega Specialty Division, Chelmsford, MA, USA) and a fiber glass back

- up filter

(SEC

- 290

- F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test substance collected

were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and

the Geometric Standard Deviation (GSD) were determined 1



### .

6.4.4. Stability monitoring

The opacity of the test atmosphere was monitored by means of a real time aerosol monitoring system

(Microdust Pro, Casella, Amherst, NH, USA). Data obtained with this system were used to illustrate

the stability of the aerosol during exposure of the animals.

6.4.5. Temperature and relative humidity

The temperature and relative humidity were measured with a humidity and temperature indicator (E+E

1 Graphs of the cumulative mass of test substance collected (percentage of total collected) against the cut points of the

impactor stages were drawn on log

- normal paper. When drawing the graphs more weight was given to the cut points where the

cumulative mass sampled was within the range of 5 to 95%. In case a linear relationship was found, the M ass Median

Aerodynamic Diameter (MMAD), the σ 84% were read from the graph. The geometric standard deviation (gsd) was calculated as

σ 84% / MMAD. The MMAD is the particle size where 50% of the particle mass is borne by particles smaller than the MMAD, the

σ 84% is the particle size where 84% of the particle mass is borne by particles smaller than the σ 84%

.- Page 11

- 

### Elektronik, Engerwitzdorf, Austria) and were recorded after the animals were placed in the

experimental set

- up and at 30 minute intervals after initiation of the exposure. The probe was inserted

in a tube mounted in one of the free animal ports of the middle section of the exposure chamber

( APPENDIX 1

, Figure 1 ) . For the exposure at 5 mg/L, the temperature of the atmosphere was

bet ween 21.1 and 21.5 o 

### C and relative humidity was between 43 and 47%. For the exposure at 1 mg/L,

the temperature of the atmosphere was between 20.9 and 21.2 o 

### C and relative humidity was between

52 and 53%.

6.5. Test system

Species Rat: Crl:WI(Han) (outbred, SPF

- Quality)

Recognised by international guidelines as the recommended test system

(e.g. OECD, EC).

Source: Charles River Deutschland, Sulzfeld, Germany.

Number of animals 5 males and 5 females (females were nulliparous and non

- pregnant) per

exposure level. Two exposure levels were used.

Age and body weight Young adult animals were selected (approximately 10 weeks old).

Animals used within the study were of approximately the same age and

body weight variation did not exceed +/

- 20% of the sex mean.

Identification Tailmark

Health inspection A health inspection was performed prior to commencement of treatment,

to ensure that the animals were in a good state of health.

6.6. Animal husbandry

Conditions

Animals were housed in a controlled environment, in which optimal conditions were considered to be

approximately 15 air changes per hour, a temperature of 21.0 ± 3.0 ° C (actual range: 19.6

- 21.9 ° C), a

relative humidity of 40

- 70% (actual range: 40

- 56%) and 12 hours artificial fluorescent light and 12

hours darkness per day.

Accommodation

Before exposure

Group housing of five animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm)

containing sterilised sawdust as bedding material ( Litalabo, S.P.P.S., Argenteuil, France) and paper as

cage

- enrichment ( Enviro

- dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom)

. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

After exposure

Group housing as described above, except that a paper sheet was introduced into the cage covering

the bedding and cage enrichment to prevent suffocation in case of bad health condition. At the end of

the Day of exposure the paper sheet was removed.

Diet

Free access to pelleted rodent diet (SM R/M

- Z from SSNIFF® Spezia ldiäten GmbH, Soest, Germany)

except during exposure to the test substance.

Water

Free access to tap water except during exposure to the test substance.

Results of analysis for each batch of diet (nutrients and contaminants), sawdust, paper and water w ere

assessed and did not reveal any findings that were considered to have affected the study integrity. All

certificates and results of analysis are retained in the archives.- Page 12

- 

### Animal husbandry on the Day of exposure

The animals were moved to the inhalation area to in order to perform the exposure. During the

exposure, there was no access to food and water. After exposure, the animals were returned their

cages which were placed in a fume cupboard for a short time period to allow test substance remnants

to evaporate. A sheet of filter paper was used to cover the bedding material to prevent suffocation in

case of bad health condition and in order to recover and to aid the clinical observations. The sheet

was removed and before the end of the exposure day, the animals were returned to the animal room.

6.7. Study design

Target concentrations were based on the cut off concentration values specified in the UN and EC

classification guidelines. Five animals of each sex were initially exposed in a limit test for 4 hours to a

target concentration of the test substance of 5 mg/L. Based on the mortality observed, f ive animals of

each sex were exposed to the next lower target concentration of 1 mg/L

.

6.8. Treatment

Fifteen minutes after the last animal was placed the generation of the test atmosphere was started.

The exposure time was 4 hours.

6.9. Observations

Mortality/Viability Twice daily. Animals showing pain, distress or discomfort, which was

considered not transient in nature or was likely to become more severe,

were sacrificed for humane reasons based on OECD guidance document

on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was

recorded as precisely as possible.

Clinical signs During exposure

Three times during exposure for mortality, behavioural signs of distress

and effects on respiration.

Clinical signs After exposure

On Day 1, one and three hours after exposure and once daily thereafter

until Day 15. The symptoms were graded according to fixed scales and

the time of onset, degree and duration were re corded:

Maximum grade 4: grading slight (1) to very severe (4)

Maximum grade 3: grading slight (1) to severe (3)

Maximum grade 1: presence is scored (1).

Body weights Days 1 (pre

- administration), 2, 8 and 15 and at death.

Necropsy The moribund animals were sacrificed by an intraperitoneal injection with

Euthasol ® (AST Farma BV, Oudewater, The Netherlands) . All animals

assigned to the study were subjected to necropsy and descriptions of all

internal macroscopic abnormalities were recorded. Particular a ttention

was given to any changes in the respiratory tract.- Page 13

- 

### 6.10. Electronic data capture

Observations/measurements in the study were recorded electronically using the following

programme(s):

− REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA): Environmental monitoring.

− TOXDATA version 8.0 Mortality / Clinical

signs / Body weights. Clinical signs during exposure or not defined in TOXDATA and body weights

of decedent animals were recorded manually

.

− Microdust Pro version 1.4 Aerosol monitoring (Casella CEL Ltd., Kempston, Bedford, UK): Test

atmosphere stability.

6.11. Interpretation

The LC 50,4h 

### value of the test substance was ranked within the following ranges:

0

– 0.05; 0. 05

– 0.5; 0.5

– 1; 1

– 5 or as exceeding 5 mg/L.

No statistical analysis was performed.

The results were evaluated according to the Globally Harmonized System of Classification and

Labelling of Chemicals (GHS) of the United Nations and Regulation (EC) No 1272/2008 of the

European Parliament and of the Council of 16 December 2008 on classification, labelling and

packaging of substances and mixtures

.

6.12. List of deviations

6.12.1. List of protocol deviations

1. Inadvertently , no body weights were determined on Day 4 f or the animals exposed to 1 mg/L.

Evaluation: Sufficient body weight data were available to evaluate the effect on body weight.

The study integrity was not adversely affected by the deviations.

6.12.2. List of standard operating procedures deviations

There were no deviations from standard operating procedures during the study.- Page 14

- 

### 7. RESULTS

7.1. Test atmosphere characterization

7.1.1. Concentration ( APPENDIX 1

, Table 1 and Figure 3 )

For the 5 mg/L expos ure group, the time

- weighted mean actual concentration was 5.1 ± 0.2 mg/L. The

nominal concentration was 4.9 mg/L. The generation efficiency (ratio of actual and nominal

concentration) was 104%. Data obtained from the opacity monitor showed that the aeroso l was

sufficiently stable.

For the 1 mg/l exposure group, the time

- weighted mean actual concentration was 1.0 ± 0.05 mg/L.

The nominal concentration was 1.15 mg/L. The generation efficiency (ratio of actual and nominal

concentration) was 87%. Data obtained from the opacity monitor showed that the aerosol was

sufficiently stable. The short drops in opacity were caused by adjustments to the generation equipment

and were considered not to have affected the exposure level.

7.1.2. Particle size ( APPENDIX 1

, Table 2 )

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were

determined twice. For the 5 mg/L exposure group, the MMAD was 2.9 μm (gsd 1.9) and 2.6 μm (gsd

2.1). For the 1 mg/L exposure group, the MMAD was 2.8 μm (gsd 2.1) and 2.9 μm (gsd 1.9).

7.2. Observations

7.2.1. Mortality ( APPENDIX 1

, Table 3 )

At 5 mg/L, on the day following exposure (Day 2) three males and two females were found dead and

the remaining animals were sacrificed for ethical reasons

. At 1 mg/L, one female was sacrificed for ethical reasons on Day 2. No further mortality occurred.

7.2.2. Clinical Signs ( APPENDIX 1

, Table 4 )

At 5 mg/L, the animals showed gasping and laboured respiration during exposure. After exposure,

hunched posture, lethargy, flat posture, laboured respiration, piloerection, ptosis and/or moribund

status were shown by the animals.

At 1 mg/L, no clinical signs were noted during exposure. After exposure, the animals showed lethargy,

hunched posture, piloerection and/or ptosis between Days 1 and 4. In addition, the female sacrificed

on Day 2 showed uncoordinated movements, chromodacryorrhoea, hypothermia, shallow respiration

and a lean appearance.

7.2.3. Body Weights ( APPENDIX 1

, Table 5 )

At 5 mg/L, body weights loss was noted in the animals found dead or sacrificed on Day 2. At 1 mg/L,

overall body weight gai n and body weight loss in males and females was within the range expected for

rats of this strain and age used in this type of study.- Page 15

- 

### 7.3. Macroscopic Findings ( APPENDIX 1

, Table 6 )

At 5 mg/L, macroscopic post mortem examination of the animals that were found dead or sacrificed for

ethical reasons during the study revealed abnormalities of the lungs (many dark red foci), stomach

(black foci in the glandular mucosa), liver (pale discoloration) and mandibular lymph nodes (dark red

discoloration). At 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the

animals.

Incidental findings included advanced autolysis which is not toxicologically relevant and pelvic dilation

of the kidneys which is occasionally seen among rats of this age and strain and therefore considered

not related to treatment.

8. CONCLUSION

The inhalatory LC 50, 4h 

### value of in Wistar rats was established to be within the

range of 1

– 5 mg/L.

Based on these results and according to the:

- Globally harmonized system of classification of chemicals (GHS), United Nations, New York and

Geneva (2011), should be classified in Class 4 for acute toxicity by the

inhalation route.

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and

mixtures, should be classified as Category 4 and should be labeled as H332:

Harmful if inhaled.- Page 16

- 

### APPENDIX 1 FIGURES AND TABLES- Page 17

- 

### Figure 1 : Schematic presentation of the experimental set

- up used for exposure



## 1. Test substance container

2. Peristaltic pump.

3. Nebulizer.

4. Pressurized dry air inlet ( à ).

5. Pressurized humidified air inlet ( à ).

6. Opacity meter.

7a. Test atmosphere inlet to animal.

7b. Exhaust outlet from animal.

8. Exposure chamber, three levels (No 1 at the top, No 3 at the bottom).

9. Animal restrainer.

10. Openings used for concentration, temperature and relative humidity

measurements.

11. Main exhaust outlet of exposure chamber to vacuum pump. 11. 10.

8.

9. 4. 7a. 7b.

1. 2. 3. 5.



## O 6.- Page 18

- 

### Figure 2 : Schematic presentation of the placement of the animals and the location of the

sampling point during exposure

5 mg/L exposure

1 mg/L exposure

1.

- 99. animal number.

c. closed atmosphere inlet to animal port.

o. open atmosphere inlet to animal port

p. pressure gauge (inlet close d).

m opening used for total concentration and for particle sizing (inlet open).

t/rh temperature and relative humidity (inlet open). 7 10 c

6 c 9

c

4 1 t/rh

8

m c

3 p

c c

c

2

Level 1

(top) Level 2 c

5 c c

Level 3 c

17 20 0

16 0 19

c

15 12 t/rh

18

m 0

13 p

c 0

0

11

Level 1

(top) Level 2 c

14 c c

Level 3 0- Page 19

- 

### Figure 3 : Stability of the test atmosphere

5 mg/L exposure

The opacity of the test atmosphere demonstrates a stable test atmosphere.



### 1 mg/L exposure

Note: The short drops in opacity were caused by adjustments to the generation equipment

and were considered not to have affected the exposure level.- Page 20

- 

### Table 1 : Gravimetrical concentration of test substance

5 mg/L exposure

Start of generation of test atmosphere: 08:23

End of generation of test atmosphere: 13:23

Time

(hh:mm) Action Sample

volume

(L) Mass

sampled

(mg) Concentration

(mg/L) % of total

expo sure time Weight

concentration

(mg/L)

08:23 start of generation n.a. n.a. n.a. 0.0 n.a.

08:26 start of sampling 5 20.86 4.172 1.3 0.052

08:31 start of sampling 5 23.67 4.734 2.1 0.099

08:36 start of sampling 5 26.45 5.290 2.1 0.110

08:50 start of samp ling 5 26.92 5.384 5.8 0.314

08:56 start of sampling 5 24.02 4.804 2.5 0.120

09:02 start of sampling 5 23.40 4.680 2.5 0.117

09:08 start of sampling 5 24.15 4.830 2.5 0.121

09:14 start of sampling 5 25.37 5.074 2.5 0.127

09:40 start of sampling 5 25.0 5 5.010 10.8 0.543

10:08 start of sampling 5 27.04 5.408 11.7 0.631

10:17 start of sampling 5 23.99 4.798 3.7 0.180

10:30 start of sampling 5 27.02 5.404 5.4 0.293

11:00 start of sampling 5 24.98 4.996 12.5 0.625

11:26 start of sampling 5 26.05 5.210 10.8 0.564

11:55 start of sampling 5 24.34 4.868 12.1 0.588

12:17 start of sampling 5 25.61 5.122 9.2 0.470

12:23 end of generation n.a. n.a. 5.122 1) 2.5 0.128

Time

- weighted mean concentration 5.081

Standard deviation 0.219

n 16



### 1 mg/L exposure

Start of generation of test atmosphere: 09:05

End of generation of test atmosphere: 13:05

Time

(hh:mm) Action Sample

volume

(L) Mass

sampled

(mg) Concentration

(mg/L) % of total

exposure time Weight

concentration

(mg/L)

09:0 5 start of generation n.a. n.a. n.a. 0.0 n.a.

09:07 start of sampling 5 0:02 1.022 0.8 0.009

09:21 start of sampling 5 0:14 1.314 5.8 0.077

09:26 start of sampling 5 0:05 0.970 2.1 0.020

09:32 start of sampling 5 0:06 1.042 2.5 0.026

09:58 start of sa mpling 5 0:26 1.060 10.8 0.115

10:26 start of sampling 5 0:28 0.998 11.7 0.116

10:57 start of sampling 5 0:31 0.994 12.9 0.128

11:23 start of sampling 5 0:26 1.038 10.8 0.112

12:04 start of sampling 5 0:41 1.012 17.1 0.173

12:31 start of sampling 5 0:27 1.042 11.3 0.117

12:56 start of sampling 5 0:25 1.022 10.4 0.106

13:05 end of generation n.a. 0:09 1.022 1) 3.8 0.038

Time

- weighted mean concentration 1.038

Standard deviation 0.052

n 11

1) Assumed concentration, based on the last sample.

2) n.a.= not applicable- Page 21

- 

### Table 2 : Aerodynamic particle size distribution in the test atmosphere

5 mg/L exposure

Start of generation of test atmosphere: 08:23

End of generation of test atmosphere: 13:23

Sampling sp eed (L/min): 2



### measurement 1:

Sampling time: 9:20

Sample volume (L): 2

Stage Cut point

( μ m) Mass sampled

(mg) Relative mass

(%) Cumulative mass

(% of total sampled)

1 21.0 0.08 0.93 99.07

2 15.0 0.05 0.58 98.48

3 10.0 0.07 0.82 97.67

4 6.0 0.47 5.48 92.19

5 3.5 2.15 25.06 67.13

6 2.0 4.44 51.75 15.38

7 0.9 0.89 10.37 5.01

8 0.5 0.43 5.01 0.00

Back up 0.25 0.00 0.00 0.00

MMAD 1 ( μ m): 2.9

gsd 2

: 1.9



### measurement 2:

Sampling time: 11:33

Sample volume (L): 2

Stage Cut point

( μ m) Mass sampled

(mg) Relative mass

(%) Cumulative mass

(% of total sampled)

1 21.0 0.09 1.20 98.80

2 15.0 0.00 0.00 98.80

3 10.0 0.02 0.27 98.54

4 6.0 0.34 4.52 94.02

5 3.5 1.98 26.33 67.69

6 2.0 3.51 46.68 21.01

7 0.9 0.86 11.44 9.57

8 0.5 0.44 5.85 3.72

Back up 0.25 0.28 3.72 0.00

MMAD 1 ( μ m): 2.6

gsd 2

: 2.1

1 Mass Median Aerodynamic Diameter

2 Geometric standard deviation- Page 22

- 

### Table 2: Aerodynamic particle size distribution in the test atmosphere (cont’d)

1 mg/L exposure

Start of generation of test atmos phere: 09:05

End of generation of test atmosphere: 13:05

Sampling speed (L/min): 2



### measurement 1:

Sampling time: 10:00

Sample volume (L): 2

Stage Cut point

( μ m) Mass sampled

(mg) Relative mass

(%) Cumulative mass

(% of total sampled)

1 21.0 0.00 0.00 100.00

2 15.0 0.00 0.00 100.00

3 10.0 0.00 0.00 100.00

4 6.0 0.09 4.66 95.34

5 3.5 0.63 32.64 62.69

6 2.0 0.84 43.52 19.17

7 0.9 0.26 13.47 5.70

8 0.5 0.07 3.63 2.07

Back up 0.25 0.04 2.07 0.00

MMAD 1 ( μ m): 2.8

gsd 2

: 2.1



### measurement 2:

Samp ling time: 11:41

Sample volume (L): 10

Stage Cut point

( μ m) Mass sampled

(mg) Relative mass

(%) Cumulative mass

(% of total sampled)

1 21.0 0.02 0.27 99.73

2 15.0 0.00 0.00 99.73

3 10.0 0.01 0.14 99.59

4 6.0 0.43 5.87 93.72

5 3.5 2.41 32.88 60.85

6 2.0 3.57 48.70 12.14

7 0.9 0.57 7.78 4.37

8 0.5 0.13 1.77 2.59

Back up 0.25 0.19 2.59 0.00

MMAD 1 ( μ m): 2.9

gsd 2

: 1.9

1 Mass Median Aerodynamic Diameter

2 Geometric standard deviation- Page 23

- 

### Table 3 : Mortality

TEST DA Y

HOURS AFTER TREATMENT 1

1 1

3 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MALES 5 MG/LITER

- - 5

- - - - - - - - - - - - - FEMALES 5 MG/LITER

- - 5

- - - - - - - - - - - - - MALES 1 MG/LITER

- - - - - - - - - - - - - - - - FEMALES 1 MG/LITER

- - 1

- - - - - - - - - - - - -



### Table 4 : Clinical signs

TEST DAY

HOURS AFTER TREATMENT MAX

GRADE 1

1 1

3 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MALES 5 MG/LITE R

ANIMAL 1

Behavior

Lethargy (3) 3 3 +

Posture

Flat posture (1) 1 1 +

Breathing

Laboured respiration (3) 2 2 +

Skin / fur

Piloerection (1) 1 1 +

Various

Ptosis (3) 3 3 +

ANIMAL 2

Behavior

Lethargy (3) 3 3 +

Posture

Flat posture (1) 1 1 +

Breathing

Laboured respiration (3) 2 2 +

Skin / fur

Piloerection (1) 1 1 +

Various

Ptosis (3) 3 3 +

ANIMAL 3

Behavior

Lethargy (3) 3 3 3 +

Posture

Flat posture (1) 1 1 1 +

Breathing

Laboured respiration (3) 2 2 3 +

Skin / fur

Piloerection (1) 1 1 1 +

Various

Moribund (1)

- - 1 +

Ptosis (3) 3 3 3 +

ANIMAL 4

Behavior

Lethargy (3) 2 3 3 +

Posture

Hunched posture (1) 1 1 1 +

Breathing

Laboured respiration (3) 2 2 3 +

Skin / fur

Piloerection (1) 1 1 1 +

Various

Ptosis (3) 2 2 2 +

ANIMAL 5

Behavior

Lethargy (3) 3 3 +

Posture

Flat posture (1) 1 1 +

Breathing

- = SIGN NOT OBSERVED / . = OBSERVATION NOT PERFORMED / + = ANIMAL DEAD- Page 24

- 

### Table 4: Clinical signs (cont’d)

TEST DAY

HOURS AFTER TREATMENT MAX

GRADE 1

1 1

3 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MALES 5 MG/LITER

Laboured respiration (3) 2 2 +

Skin / fur

Piloerection (1) 1 1 +

Various

Ptosis (3) 2 2 +

FEMALES 5 MG/LITER

ANIMAL 6

Behavior

Lethargy (3) 3 3 +

Pos ture

Flat posture (1) 1 1 +

Breathing

Laboured respiration (3) 2 2 +

Skin / fur

Piloerection (1) 1 1 +

Various

Ptosis (3) 3 3 +

ANIMAL 7

Behavior

Lethargy (3) 3 3 +

Posture

Flat posture (1) 1 1 +

Breathing

Laboured respiration (3) 2 2 +

Skin / fur

Piloerection (1) 1 1 +

Various

Ptosis (3) 3 3 +

ANIMAL 8

Behavior

Lethargy (3) 3 3 3 +

Posture

Flat posture (1) 1 1 1 +

Breathing

Laboured respiration (3) 2 2 3 +

Skin / fur

Piloerection (1) 1 1 1 +

Various

Moribund (1)

- - 1 +

Ptosis (3) 3 3 3 +

ANIMAL 9

Behavior

Lethargy (3) 3 3 3 +

Posture

Flat posture (1) 1 1 1 +

Breathing

Laboured respiration (3) 2 2 3 +

Skin / fur

Piloerection (1) 1 1 1 +

Various

Moribund (1)

- - 1 +

Ptosis (3) 3 3 3 +

ANIMAL 10

Behavior

Lethargy (3) 3 3 3 +

Posture

Flat posture (1) 1 1 1 +

Breathing

Laboured respiration (3) 2 2 3 +

Skin / fur

Piloerection (1) 1 1 1 +

Various

Moribund (1)

- - 1 +

Ptosis (3) 3 3 3 +

- = SIGN NOT OBSERVED / . = OBSERVATION NOT PERFORMED / + = ANIMAL DEAD- Page 25

- 

### Table 4: Clinical signs (cont’d)

TEST DAY

HOURS AFTER TREATMENT MAX

GRADE 1

1 1

3 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MALES 1 MG/LITER

ANIMAL 11

Posture

Hunched posture (1) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerection (1 ) 1 1

- - - - - - - - - - - - - -

ANIMAL 12

Behavior

Lethargy (3) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1 ) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerection (1) 1 1

- - - - - - - - - - - - - -

ANIMAL 13

Behavior

Lethargy (3 ) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerection (1) 1 1

- - - - - - - - - - - - - -

Various

Ptosis (3) 1

- - - - - - - - - - - - - - -

ANIMAL 14

Behavior

Lethargy (3) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1 1

- - - - - - - - - - - Skin / fur

Piloerection (1) 1 1

- - - - - - - - - - - - - -

ANIMAL 15

Behavior

Lethargy (3) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerec tion (1) 1 1

- - - - - - - - - - - - - - FEMALES 1 MG/LITER

ANIMAL 16

Behavior

Lethargy (3) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerection (1) 1 1

- - - - - - - - - - - - - -

ANIMAL 17

Behavior

Lethargy (3) 1 1 3 +

Posture

Hunched posture (1)

- 1 1 + Gait / motility

Uncoordinated movements (3)

- - 1 +

Breathing

Shallow respiration (3)

- - 1 +

Skin / fur

Piloerection (1) 1 1 1 +

Secretion / excretion

Chromodacryorrhoea (Snout) (3)

- - 1 +

Various

Lean (1)

- - 1 +

Ptosis (3)

- - 1 +

Hypothermia (1)

- - 1 +

- = SIGN NOT OBSERVED / . = OBSERVATION NOT PERFORMED / + = ANIMAL DEAD- Page 26

- 

### Table 4: Clinical signs (cont’d)

TEST DAY

HOURS AFTER TREATMENT MAX

GRADE 1

1 1

3 2 3 4 5 6 7 8 9 10 11 12 13 14 15

FEMALES 1 MG/LITER

ANIMAL 18

Behavior

Lethargy (3) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerection (1) 1 1

- - - - - - - - - - - - - -

ANIMAL 19

Behavior

Lethargy (3) 1 1

- - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1 1

- - - - - - - - - - - Skin / fur

Piloerection (1) 1 1 1

- - - - - - - - - - - - - ANIMAL 20

Behavior

Lethargy (3) 1

- - - - - - - - - - - - - - -

Posture

Hunched posture (1) 1 1 1 1

- - - - - - - - - - - - Skin / fur

Piloerection (1) 1 1

- - - - - - - - - - - - - -

- = SIGN NOT OBSERVED / . = OBSERVATION NOT PERFORMED / + = ANIMAL DEAD- Page 27

- 

### Table 5 : Bod y weights (gram)

SEX/DOSE LEVEL ANIMAL DAY 1 DAY 2 DAY 8 DAY 15

MALES 5 MG/LITER

1 280 257 *

--- ---

2 267 248 *

--- ---

3 282 254 **

--- ---

4 288 258 **

--- ---

5 276 262 *

--- ---

MEAN 279

--- --- ST.DEV. 8

--- ---

N 5 0 0

FEMALES 5 MG/LITER

6 194 168 *

--- ---

7 197 185 *

--- ---

8 188 172 **

--- ---

9 184 168 **

--- ---

10 213 191 **

--- ---

MEAN 195

--- --- ST.DEV. 11

--- ---

N 5 0 0

MALES 1 MG/LITER

11 270 260 280 299

12 299 286 311 333

13 283 271 290 310

14 295 285 301 320

15 290 282 308 322

MEAN 287 277 298 317

ST.DEV. 11 11 13 13

N 5 5 5 5

FEMALES 1 MG/LITER

16 190 187 199 208

17 216 181 **

--- ---

18 200 196 210 218

19 199 194 208 217

20 191 181 194 203

MEAN 199 188 203 212

ST.DEV. 10 7 8 7

N 5 5 4 4



### *. Animal found dead

**. Animal sacrificed for humane reasons

Note: No mean was calculated for Day 2 at 5 mg/L, since some animals were weighed after being found dead.- Page 28

- 

### Table 6 : Macroscopic findings

ANIMAL ORGAN FINDING DAY OF DEATH

MALES 5 MG/LITER

1 General observations Advanced autolysis. Spontaneous death

Lungs Focus/foci, many, dark red. Day 2 after treatment

2 General observations Advanced autolysis. Spontaneous death

Lungs Focus/foci, many, dark red. Day 2 after treatment

3 Stomach Glandular mucosa: focus/foci, several, Killed in extremis

Black. Day 2 after treatment

Liver Discol ouration, pale.

4 Stomach Glandular mucosa: focus/foci, several, Killed in extremis

Black. Day 2 after treatment

Liver Discolouration, pale.

Mandibular lymph n Both sides: discolouration, dark red.

5 General observation s Advanced autolysis. Spontaneous death

Lungs Focus/foci, many, dark red. Day 2 after treatment

FEMALES 5 MG/LITER

6 General observations Advanced autolysis. Spontaneous death

Lungs Focus/foci, many, dark red. Day 2 after treatment

7 General observations Advanced autolysis. Spontaneous death

Lungs Focus/foci, many, dark red. Day 2 after treatment

8 Stomach Glandular mucosa: focus/foci, several, Killed in extremis

Black. Day 2 after treatment

Liver Discol ouration, pale.

9 Stomach Glandular mucosa: focus/foci, Killed in extremis

Isolated, black. Day 2 after treatment

10 Kidneys Right side: pelvic dilation. Killed in extremis

Day 2 after treatment

MALES 1 MG/LITER

11 No findings noted Scheduled necropsy

Day 15 after treatment

12 No findings noted Scheduled necropsy

Day 15 after treatment

13 No findings noted Scheduled necropsy

Day 15 after treatment

14 No findings noted Scheduled necropsy

Day 15 after treatment

15 No findings noted Scheduled necropsy

Day 15 after treatment

FEMALES 1 MG/LITER

16 No findings noted Scheduled necropsy

Day 15 after treatment

17 No findings n oted Killed in extremis

Day 2 after treatment

18 No findings noted Scheduled necropsy

Day 15 after treatment

19 No findings noted Scheduled necropsy

Day 15 after treatment

20 No findings noted Scheduled necro psy

Day 15 after treatment