FINAL REPORT 

Study Title

ASSESSMENT OF ACUTE INHALATION TOXICITY WITH

IN THE RAT

Author

Test Facility

Laboratory Project Identification

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1. CONTENTS

1. CONTENTS ...................................................................................................................................... 2

2. STATEMENT 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 study............................................................................................................................ 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 characterization ........................................................................................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 (APPENDIX 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

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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), L142, 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%. Data 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 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. 

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 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.

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CONCLUSION

The inhalatory LC50, 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. 

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5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Study 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, 

May 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, 

April 2011, including the most recent partial revisions. 

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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. 

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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 container 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.

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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 total 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 Mass Median 

Aerodynamic Diameter (MMAD), the σ84% were read from the graph. The geometric standard deviation (gsd) was calculated as 

σ84% / MMAD. The MMAD is the particle size where 50% of the particle mass is borne by particles smaller than the MMAD, the 

σ84% is the particle size where 84% of the particle mass is borne by particles smaller than the σ84%. 

Final Report
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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 

between 21.1 and 21.5o

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 21.2o

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® Spezialdiäten 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 were 

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.

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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, five 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 recorded:

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 attention 

was given to any changes in the respiratory tract. 

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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 LC50,4h 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 for 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.

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7. RESULTS

7.1. Test atmosphere characterization

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

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%. Data 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. 

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 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.

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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 LC50, 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. 

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APPENDIX 1 FIGURES AND TABLES

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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.

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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 closed).

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

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

7 c 10

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 0 20

16 0 19

c

15 12

t/rh

18

0 m

13

 p

 c

 0

 0

11

Level 1

(top)

Level 2

c 

14

 c

c

Level 3

0

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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. 

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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 

exposure 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 sampling 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 25.05 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)

09:05 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 sampling 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

Final Report
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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 speed (L/min): 2

measurement 1:

Sampling time: 9:20

Sample volume (L): 2

Stage Cut point

(μm)

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

MMAD1 (μm): 2.9

gsd2: 1.9

measurement 2:

Sampling time: 11:33

Sample volume (L): 2 

Stage Cut point

(μm)

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

MMAD1 (μm): 2.6

gsd2: 2.1 1

 Mass Median Aerodynamic Diameter 2

 Geometric standard deviation

Final Report
- Page 22 - 

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

1 mg/L exposure

Start of generation of test atmosphere: 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

(μm)

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

MMAD1 (μm): 2.8

gsd2: 2.1

measurement 2:

Sampling time: 11:41

Sample volume (L): 10

Stage Cut point

(μm)

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

MMAD1 (μm): 2.9

gsd2: 1.9 1

 Mass Median Aerodynamic Diameter 2

 Geometric standard deviation

Final Report
- Page 23 - 

Table 3: Mortality 

 

TEST DAY 

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/LITER 

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

Final Report
- 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 +

Posture 

 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 

Final Report
- 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 

 Piloerection (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 

Final Report
- 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 

Final Report
- Page 27 - 

Table 5: Body 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.

Final Report
- 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 Discolouration, 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 observations 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 Discolouration, 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 noted 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 necropsy

 Day 15 after treatment

Final Report