### TITLE PAGE

STUDY TITLE

Report

Cobalt Lithium Manganese Nickel oxide

- Short

- term (5

- day ) inhalation study in Wistar rats dust exposure

TEST GUIDELINE(S) No testing guidelines exist for this type of study

AUTHOR(S)

STUDY COMPLETION DATE
12 Aug 2013

TEST FACILITY

TEST FACILITY PROJECT IDEN TIFICATION

SPONSOR

PART I OF III (REPORT SECTION AND SUMMARY TABLES)

This document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be use d only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, pub lication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory ag encies acting within the limits of their administrative authority.



### Page 1 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors **The use of these studies/documents for regulatory, registration or other commercial purposes, including but not limited to Registration, Evaluation, Authorization and Restriction of Chemical Substances (REACH), without**  **the express written permission of the sponsor is unauthorized and strictly prohibited.** 

### EPA-HQ-OPPT-2009-0922 SANITIZED

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### CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATO RY PERSONNEL

Head of Experimental Toxicology and Ecology:

Study director:

Clinical Pathology:

Pathology:

Data processing:

Statistics:

Analytical Chemistry:

Quality Assurance Unit (QAU):

Coordination QAU:

Inhalation Toxicology:

Clinical Chemistry:

Pathology:

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### GLP CERTIFICATE (FROM THE COMPETENT AUTH ORITY)

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### CONTENTS

TITLE PAGE
STATEMENT OF NO DATA CONFIDENTIALITY CLAI MS
BLANK PAGE FOR EPA SUBMISSION ONLY FLAGGING CRITERIA
SIGNATURE PAGE
CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATO RY PERSONNEL STATEMENT OF THE QUA LITY ASSURANCE UNIT
GLP CERTIF ICATE (FROM THE COMP ETENT AUTHORITY)
CONTENTS
1. SUMMARY
2. INTRODUCTION
2.1. OBJECTIVES
2.2. SELECTION OF CONCENT RATIONS
2.3. TEST GUIDELINES
2.4. TIME SCHEDULE
2.5. RETENTION OF RECORDS
2.6. ANIMAL WELFARE
3. MATERIALS AND METHOD S
3.1. TEST ITEM
3.2. TEST SYSTEM
3.3. HOUSING AND DIET
3.4. TEST GROUPS AND TARGET CO NCENTRATIONS
3.5. GENERATION OF THE IN HALATION ATMOSPHERES
3.6. ANALYSES
3.6.1. Calculation of nominal concentrations
3.6.2. Determination of atmospheric concentrations
3.6.2.1. Sampling for gravimetric analyses
3.6.2.2. Gravimetrical method of analyses
3.6.2.3. Real time monitoring of constancy of concentrations
3.6.3. Particle size analysis
3.6.3.1. Definitions
3.6.3.2. Equipment, sampling and method of determination
3.6.3.3. Particle size analysis by Aerodynamic Particle Sizer (APS)
3.6.4. Food analyses
3.6.5. Drinking water analyses
3.6.6. Bedding and enrichment analyses
3.7. EXPERIMENTAL PROCEDU RE

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### 3.7.1. Acclimatization and exposure period
3.7.2. Exposure systems; exposure of the animals
3.7.3. Measurements of the exposure conditions
3.8. CLINICAL EXAMINATION S
3.8.1. Mortality
3.8.2. Clinical observations
3.8.3. Body weight data
3.8.4. Urine collection
3.8.5. Rectal temperature
3.9. LUNG BURDEN
3.10. CLINICAL PATHOLOGY
3.10.1. Hematology
3.10.2. Clinical chemistry
3.10.3. Acute phase proteins in serum
3.10.4. Bronchoalveolar lavage fluid (BAL)
3.10.4.1. Cytology in BAL
3.10.4.2. Total Protein and enzymes in BAL
3.11. PATHOLOGY
3.11.1. Necropsy
3.11.2. Weight parameters
3.11.3. Organ / Tissue preservation list
3.11.4. Histopathology
3.12. STATISTICS
4. RESULTS
4.1. ANALYSES
4.1.1. Measurements of atmosphere parameters and operation conditions
4.1.1.1. Concentration measurements in the exposure system
4.1.1.2. Measurements concerning operation conditions
4.1.1.3. Results of the particle size analyses
4.1.2. Food analyses
4.1.3. Drinking water analyses
4.1.4. Bedding and enrichment analyses
4.2. CLINICAL EXAMINATION S
4.2.1. Mortality
4.2.2. Clinical observations
4.2.3. Body weight data
4.2.4. Rectal temperature
4.2.5. Urinary excretion of metal ions
4.3. ORGAN BURDEN

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### 4.4. CLINICAL PATHOLOGY
4.4.1. Hematology
4.4.2. Clinical chemistry
4.4.3. Acute phase proteins
4.4.4. Bronchoalveolar lavage fluid (BAL)
4.4.4.1. Cytology in BAL
4.4.4.2. Total Protein and enzymes in BAL
4.5. PATHOLOGY
4.5.1. Weight parameters
4.5.2. Gross lesions
4.5.3. Histopathology
5. DISCUSSION
6. CONCLUSION
7. REFERENCES
8. LIST OF ABBREVIATION S
8.1. CLINICAL EXAMINATION S
8.2. ANALYTICAL DATA AND EXPOSURE PARAMETERS
8.3. CLINICAL PATHOLOGY
8.4. PATHOLOGY

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### INCIDENCE AND SUMMARY TABLES

CLINICAL EXAMINATIONS (Mean values and Summary tables) Table

Summary - Clinical Observation Male animals IA -1 – IA -3

Summary Body Weights Male animals IA

- 4 – IA -5

Summary Changes B ody W eight s Male animals IA -6

Rectal Temperature Male animals IA -7

CLINICAL PATHOLOGY (Mean values and Summary tables)

Groups 0

- 2 (nominal days 7 + 28)

Red blood cell + coagulation parameters IB

- 1 – IB -2 White blood cell parameters IB

- 3 – IB -4 Enzymes IB

- 5 – IB -6 Substrates IB

- 7 – IB -8 Electrolytes + minerals IB

- 9 – IB

- 10 Acute phase proteins IB

- 11 – IB -12 BAL: cytology IB

- 13 – IB

- 16 BAL: enzymes + protein IB

- 17 – IB

- 18

Groups 0+3 (nominal day 2)

Red blood cell + coagulation parameters IB

- 19 White blood cell parameters IB

- 20 Enzymes IB

- 21 Substrates IB

- 22 Electrolytes + minerals IB

- 23

Groups 0+3 (nominal day 25)

Red blood cell + coagulation parameters IB

- 24 White blood cell parameters IB

- 25 Enzymes IB

- 26 Substrates IB

- 27 Electrolytes + minerals IB

- 28

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### PATHOLOGY (Mean values and Summary tables)

Absolute weights - mean values (male), (main group s) IC

- 1 - IC -2 Relative weights - mean values (male), (main groups) IC -3 - IC -4 Absolute weights - mean values (male), (recovery groups) IC

- 5 - IC -6 Relative weights - mean values (male), (recovery groups) IC

- 7 - IC -8 Incidence of gross lesions (main groups) IC -9 Incidence of gross lesions (recovery groups) IC

- 10 Incidence of gross lesions (premature death) IC

- 11 Incidence of all microscopic findings IC -1 2 – IC

- 14 Incidence and grading of microscopic findings IC

- 15 - IC -18

MEASUREMENTS OF EXPOSURE PARAMETERS (Summary table)

Exposure P arameters ID -1

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### PART II: INDIVIDUAL TABLES AND FIGURES

CLINICAL EXAMINATIONS (Individual values) Table

Individual Signs By Interval – Clinical Observation Male animals IIA

- 1 - IIA

- 11

Individual Body W eigh ts Male animals IIA -12 - IIA

- 15

Individual Rectal Temperature Male animals IIA

- 16 - IIA

- 19

CLINICAL PATHOLOGY (Individual values)

Groups 0

- 2 (study days 7 + 28)

Red blood cell + coagulation parameters IIB

- 1 – IIB -2 White blood cell parameters IIB

- 3 – IIB -4 Enzymes IIB

- 5 – IIB -6 Substrates IIB

- 7 – IIB -8 Electrolytes + minerals IIB

- 9 – IIB

- 10 Acute phase proteins IIB

- 11 – IIB

- 12 BAL: cytology IIB

- 13 – IIB

- 16 BAL: enzymes + protein IIB

- 17 – IIB

- 18

Groups 0+3 (study day 2)

Red blood cell + coagulation pa rameters IIB

- 19 White blood cell parameters IIB

- 20 Enzymes IIB

- 21 Substrates IIB

- 22 Electrolytes + minerals IIB

- 23

Groups 0+3 (study day 25)

Red blood cell + coagulation parameters IIB

- 24 White blood cell parameters IIB

- 25 Enzymes IIB

- 26 Substrates IIB

- 27 Electrolytes + minerals IIB

- 28

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### PATHOLOGY (Individual values)

Absolute weights IIC

- 1 - IIC -14 Relative weights IIC

- 15 - IIC -28 Single animal sheet IIC

- 29 - IIC

- 53 (Gross lesions and microscopic findings)

MEASUREMENTS OF EXPOSURE PARAMETERS

Exp osure parameters (Daily Means) IID

- 1 - IID -4 Particle size analysis (Individual measurements) IID

- 5 - IID -9

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### PART III: SUPPLEMENT

Analysis of the test substance

Characterization of the test substance

Historical control data of clinical pathology testing

Analysis of urine samples

Analysis of lungs

Analysis of extra

- pulmonary organs

Analysis of blood

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## 1. SUMMARY

The purpose of this study was to determine the pulmonary toxicity in rats using a short

- term bioassay including bronchoalveolar lavage with clinico

- chemical and cytological evaluation of lavage fluid and pathological examination of the respiratory tract . The results of this study should facilitate the concentration selection of a following repeated dose inhalation study. For this study, milled material was used to enable dust generation.

Groups of male Wistar rats were exposed nose

- only to the dust aerosol of the test item for 6 hours per day on 5 consecutive days. The target concentrations are 2, 10 and 50 mg/m 3 

### . A concurrent control group is exposed to clean air. Daily clinical observations, body weight were recorded. Additional assessments including clinical chemistry, hematology, and histopathology were carried out at the termination of the study.

Results:

A s shown in table 1, the targeted atmospheric concentrations were maintained well throughout the whole study. Control animals were exposed to conditioned air. The following study means of concentrations were determined by gravimetrical analysis

.

Table 1 : Study means and standard deviations of test substance concentrations and particle size distribution

Test
group Target concentration (mg/m3) Measured concentration (mg/m3) Mass median aerodynamic diameter (μm) Mean SD 1 2 2.2 0. 6 3.2 and 3. 8
2 10 10

. 0 1

. 9 3.4 and 3

. 4
3 5 0 5 4

. 5 4

. 0 4.9

The following substance

- related adverse effects were observed:

Test group 3 (5 0 mg/m3 )

• 16 of the total 22 animals found dead or sacrificed moribund on study day 2 and 3 (after 2 exposures)

• Clinical signs of toxicity comprised accelerated respiration, ruffled fur, hypothermia

, poor general condition and reduced body weight.

• Premature death with acute alveolar damage (fibrinous hyaline alveolar membranes, eosinophil degranulation and type II pneumocyte proliferation, inflammation) in the lungs

• Degeneration/ regeneration of the bronchial epithelium of the lungs

• Degeneration of the olfactory epithelium level III of the nasal cavity

• Necrosis of the olfactory epithelium and squamous metaplasia of the olfactory epithelium in level IV of the nasal cavity

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### • Minimal to moderate squamous metaplasia in the larynx, level I and II

• Degenerati on/regeneration of the respiratory epithelium in the trachea

Test group 3 (50 mg/m3, 2 exposures and 3 weeks recovery)

• Minimal bronchial epithelial degeneration and regeneration in the lungs

Test group 2 (10 mg/m3 on study day 7)

• Increased red blood cell (RBC) counts, hemoglobin and h aematoc rit values

• Increased alkaline phosphatase (ALP) activities in blood

• Increased total cell counts in BALF as well as absolute and relative neutrophil, lymphocyte monocyte, eosinophil and atypical cell counts

• Increased total protein in BALF as well as alkaline phosphatase (ALP), lactate dehydrogenase (LDH), γ - glutamyl transferase (GGT) and β -N- acetyl glucosaminidase (NAG) activities

• Minimal to slight bronchial epithelial degeneration and regeneration and peribronchial inflammation in the lungs in all animals

• Necrosis of the olfactory epithelium in all animals in level IV of the nasal cavity

• Degeneration/regeneration of the respiratiory epithelium in the trachea in all animals

Test group 2 (10 mg/m3 on study day 28 )

• No adverse effect observed

Test group 1 (2 mg/m3 on study day 7)

• Increased red blood cell (RBC) counts, hemoglobin and h aematoc rit values

• Increased total cell counts in BALF as well as absolute and relative neutrophil, lymphocyte monocyte, eosinophil and atypical cell counts

• Increased total protein in BALF as well as alkaline phosphatase (ALP), lactate dehydrogenase (LDH), γ - glutamyl transferase (GGT) and β -N- acetyl glucosaminidase (NAG) activities

• Minimal to slight bronchial epithelial degeneration and regeneration in the lungs in all animals

• Necrosis of the olfactory epithelium of the nasal cavity, level IV in 2/3 animals

• Degeneration/regeneration of the respiratiory epithelium in the trachea in all animals

Test group 1 (2 mg/m3 on study day 28)

• No adverse effect observed

Conclusion Inhalation exposure of 50 mg/m 3 

### Cobalt Lithium Manganese Nickel oxide caused lethality after only two exposures. Upon which , the exposure was terminated in this group . The surviving animals showed transient body weight loss, labored and intermittent respiration as well as several unspecific clinical signs of toxicity. No substance

- related adverse effect was observed in clinical pathology. Histological examination of the premature decedents showed lesions throughout the respiratory tract. Apart from minimal degeneration and regeneration of

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### the bronchial epithelia, no other morphological changes were observed in surviving animals of test group 3 sacrificed on study day 25. At 2 and 10 mg/m3 clearly treatment

- related changes were observed in lavage fluid, hematology and in histology of the respiratory tract. The effects in hematology and lavage fluid were fully reversible within 3 weeks recovery period.

Due to findings in hematology, lavage fluid and histology of the respiratory tract, A No Observed Adverse Effect Concentration (NOAEC) could not be established under the current study condition.

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## 2. INTRODUCTION

2.1. OBJECTIVES

The purpose of this study was to determine the pulmonary toxicity of Cobalt Lithium Manganese Nickel oxide in rats using a short

- term bioassay including bronchoalveolar lavage with clinico

- chemical and cytological evaluation of lavage fluid and pathological examination of the respiratory tract

.
Moreover, to examine whether certain metal ion may leach out of the solid material and become systemic ally available, urine sample of the high concentration group animals were collected after the last exposure. Lung burden of the test material was determined in sate llite animals.

The results of this study should be used to choose appropriate test concentrations in the following 90

- day inhalation study.

2.2. SELECTION OF CONCENT RATIONS

As a range finding study with a potentially poorly soluble particular matter with expected low pulmonary toxicity the concentration s are chosen based on available data of pigmentary TiO 2 

### . The following concentrations were selected for the present study:

50 mg/m3: as the high concentration expected to caus e adverse effect

10 mg/m3: as the mid concentration expected to caus e some effect 2 mg/m3: as the low concentration and the expected no adverse effect concentration

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### 2.3. TEST GUIDELINES

No testing guidelines exist for this type of study.

The conduct of inhalation exposures was performed according to the following test guidelines concerning repeated dose inhalation toxicity studies:

• Organization for Economic Cooperation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, No. 412, "Repeated Dose Inhalation Toxicity: 28

- day or 14

- day Study"

• Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Repeated dose (28 days) toxicity (inhalation) No. L142

2.4. TIME SCHEDULE

Test dates: Study initiation date: 19 Sep 201 1 Experimental starting date (Arrival of test animals): 06 Sep 201 1

Experimental completion date (check the raw data for its completeness): 07 Aug 2013

In the following table the time schedule of the study is presented. This time schedule was modified due to premature death of animals of the high concentration group.

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### Table 2 : S tudy time table

Date Study day Study section / Examination 06 Sep 2011

- 13 Arrival of the animals (age: 7 weeks); experimental starting date 15 Sep 2011 -4 Start of pre

- exposure 19 Sep 2011 0 Start of exposure period

21 Sep 2011 2 Termination of exposure for test group 3, start of recovery period of the surviving animals of test group 3

21 Sep 2011 2 Blood sampling of 5 surviving animals of test group 3 and 5 control animals Transfer of the same 5 surviving animals of test group 3 to metabolism cages. Urine is sampled.

23 Sep 2011 4 End of exposure period. Necropsy; start of pathological examination and examination of organ burden of the main group animals. 1st urine collection in recovery group animals (control and test group 2 only)

26 Sep 2011 7 Blood sampling*, lung lavage; start of clinical pathological examination of the main group animals, start of examination of urine

06 Oct 2011 17 2nd urine collection in recovery group animals (control and test group 2 only), start of examination of urine

14 Oct 2011 25 Blood sampling surviving animals of the test group 3 and 5 control animals necropsy*; start of pathological examination of the recovery group animals.

17 Oct 2011 28 Blood sampling*, lung lavage; start of clinical pathological examination of the recovery group animals.

Oct 2011 – Dec 2011 - Organ fixation, processing and evalua tion

Feb 2012 - Draft report: Clinical Pathology and Pathology (to study director)

Feb 2012 - Proposed experimental completion date; check the raw data for its completeness

March 2012 - Proposed finalization of the report

* = Food will be withdrawn from the animals overnight (about 16

- 20 hours)



### .

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### 2.5. RETENTION OF RECORDS

All r elevant records and materials are stored at or at least the period of time specified in the GLP principles. Details concerning responsibilities or locations of archiving can be seen from the respective SOPs and from the raw data.

All samples (blood samples) for clinical examination were disposed two years after the finalization of the study.

All samples (urine samples) for clinical examination were disposed three years after the finalization of the study.

2.6. ANIMAL WELFARE

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## 3. MATERIALS AND METHOD S

3.1. TEST ITEM

The technical material provided by the sponsor was coarse. No dust aerosol could be generated. Therefore, the technical material was micronized to achieve respirable particles. The test item (= test substance) refer s to the micronized material. The micronized material analyses were characterized at

Name of test substance: Cobalt Lithium Manganese Nickel oxide

Test substance No.:

Batch No.:

CAS No.: 53306

- 54 -0

Purity: 100 %

Homogeneity: Given

Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the
sponsor , and the sponsor holds this responsibility.

ADDITIONAL TEST SUBSTANCE INFORMATION

Synonyms: Cobalt Lithium Manganese Nickel oxide (milled)

Physical state/ Appearance: Solid / black

Storage conditions: Room temperature

The Sponsor is responsible for compliance for all test substance information and their storage, except for those test substance investigations commissioned by the test facility.

More details of the test substance can be found in the Part III of this report and in the raw data.

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### 3.2. TEST SYSTEM

Test species and strain: Wistar rats, Crl:WI(Han)

Reason for selection of the test species: Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Age when supplied; sex: about 7 weeks, male
Supplier: Charles River Laboratories,

Arrival in the testing facility: 06 Sep 201 1

Identification: The animals were identified individually by tattooing the respective animal number into the ears

Only animals free from clinical signs of disease were used for the study.

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### 3.3. HOUSING AND DIET

The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H- Temp [ PSU ]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm 2 

### ). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust- free wooden bedding was used in this study (the present supplier is documented in the raw data)

. For enrichment wooden gnawing blocks ( Typ NGM E

- 022), supplied by Abedd ® 

### Lab. and Vet. Service GmbH, Vienna, Austria, were added.

The animals were kept in fully air

- conditioned rooms in which a temperature in the range of 20 - 24°C and relative humidity in the range of 30 - 70% were ensured b y means of a central air

- conditioning system. 15 air changes per hour.

A light/dark rhythm of 12 hours was maintained:

• 06.00 a.m. - 06.00 p.m. light

• 06.00 p.m. - 06.00 a.m. dark

Deviations f rom these ranges did not occur. The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin

ammonia

- based terminal disinfector) before the start of the study. The floor and the walls were cleaned once a week with water containing an appropriate disinfectan t.

The animals were maintained on mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) and tap water ad libitum.

Details of the feed batch used are available in the raw data.

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### 3.4. TEST GROUPS AND TARG ET CONCENTRATIONS

Test groups, target concentrations and allocation of animals in the groups 0, 1 and 2 are presented in the tables 3 to 8

. The allocation of test group 3 animals to different examination endpoints are presented in table 9. D ue to premature death occurred in test group 3 , the initial allocation had to be modified.

Table 3 : Main groups for blood sampling and bronchoalveolar lavage

Test group Concentration (mg/m 3 ) 1 No. of animals Animal No.

0 0 5 1

- 5
1 2 5 16

- 20
2 10 5 31

- 35



### Table 4 : Main group animals for light microscopic examination

Test group Concentration (mg/m 3 ) 1 No. of animals Animal No.

0 0 3 6

- 8
1 2 3 21

- 23
2 10 3 36

- 38



### Table 5 : Main group animals for organ burden

Test group Concentration (mg/m 3 ) 1 No. of animals Animal No.

0 0 3 9

- 11
1 2 3 24

- 26
2 10 3 39

- 41



### Table 6 : Recovery group animals for blood sampling, urine collection and bronchoalveolar lavage

Test group Concentration (mg/m 3 ) 1 No. of animals Animal No.

0 0 5 76

- 80
1 2 5 91

- 95
2 10 5 106

- 110



### Table 7 : Recovery group animals for light microscopic examination

Test group Concentration (mg/m 3 ) 1 No. of animals Animal No.

0 0 3 81

- 83
1 2 3 96

- 98
2 10 3 111

- 113

1 test substance concentration



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### Table 8 : Recovery group animals for organ burden

Test group Concentration (mg/m 3 ) 1 No. of animals Animal No.

0 0 3 84

- 86
1 2 3 99

- 101
2 10 3 114

- 116



### Table 9 : Test group 3 animals and their allocation to different examination endpoints

Animal No. Blood sampling and Urine collection on study day 2 Gross necropsy* Lung burden Histological examination Sacrificed on study day 25** (incl. gross
necropsy, organ weight) 46 x x 47 a 

### x 48 a 

### x 49 a 

### x 50 x x 51 x x x 52 b 

### x x 53 x x x 54 a 

### x 55 a 

### x 56 a 

### x 121 a 

### x x 122 c 

### x x 123 x 124 c 

### x x 125 a 

### x x 126 a 

### x x 127 a 

### x x 128 a 

### x x 129 x 130 b 

### x 131 b 

### x x
** Blood sampling (hematology and clinical chemistry according to study plan), gross necropsy and histological examination according to study plan

a Found dead on study day 2
b Found dead on study day 3

c S acrificed moribund on study day 3, necropsied as soon as possible



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### 3.5. GENERATION OF THE IN HALATION ATMOSPHERES

Generator systems:

• Solid particle generators (brush

- generator; )

• Mixing tube

Generation procedure:

The test substance was used unchanged.

For each concentration the dust aerosol was generated with the dust generator and
compre ssed air inside a mixing tube mixed with conditioned dilution air and passed into the inhalation system. Conditioned supply air is activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C. Compressed air is filtered air pressurized to about 6 bar.

The following substance flow and air flows were scheduled:

Table 10 : Scheduled exposure parameters

Test
group Substance flow (mg/h) Supply air compressed (m3/h) Supply air conditioned (m3/h) Exhaust air (m3/h)

0

- - 5. 7

– 6

. 3 5

. 1

- 5

. 7 1 20

– 100 1

. 2

- 1

. 8 4.2

– 4. 8 5

. 1

- 5

. 7 2 80

– 2 00 1

. 2

- 1

. 8 4. 2

– 4

. 8 5

. 1

- 5

. 7 3 400

– 1200 1

. 2

- 1

. 8 4.2

– 4. 8 5

. 1

– 5

. 7 * conditioned air to the mixing tube, part of dilution system # air out of the mixing tube, part of dilution system



### Figure 1: Generator system

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### 3.6. ANALYSES

3.6.1. Calculation of nominal concentrations

T he nominal concentration was calculated from the study means of the substance flow and the supply air flows used during exposure to generate the respective concentrations.

3.6.2. D etermination of atmospheric concentrations

Principle:

The concentrations of the inhalation atmospheres were analyzed by gravimetry in test groups 1 - 3

.

Details were described in Part II.

Daily means were calculated based on the measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.

In these groups, the constancy of concentrations in the inhalation system s we re continuously monitored using scattered light photometers.

The analyses were carried out at the

3.6.2.1. Sampling for gravime tric analyses

Equipment

• Internal probe diameter: 7 mm

• Filter: MN 85/90 BF (d = 4.7 cm)

• Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA )

• Balance: Mettler AT 250 ( Mettler

- Toledo AG, Greifensee, Switzerland )

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### Sampling:

• Sampling velocity:

• Flow rate of sampling:

• Sample volumes:

• Test group 1:

• Test group 2:

• Test group 3: 1.25 3

360 90 18 m/s L/min

L L L

• Sampling site:

• Sampling frequency: immediately adjacent to the animals' noses at a separate spare port two samples per exposure and concentration group

3.6.2.2. Gravimetrical method of analyses

A preweighed filte r was placed into the filter holder. By means of a vacuum compressed air pump a defined volume of the dust was drawn through the filter.

The dust concentration in mg /m3 was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling , w ith reference to the sample volume of the inhalation atmosphere.

3.6.2.3. Real time monitoring of constancy of conce ntrations

Scattered light photometers (VisGuard

, Sigrist

, Ennetbürgen , Switzerland ) in test groups 1

- 3 were used to continuously monitor the constancy of concentrations of test substance dusts in the inhalation systems. The measurements were by the automated measuring system.

3.6.3. Particle size analysis

3.6.3.1. Definitions

• EACD 50%(effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages.

• MMAD(mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass.

• Geometrical standard deviation (GSD) is the ra tio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.

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### 3.6.3.2. Equipment, sampling and method of determination

The particle size analysis was carried out with a cascade impactor.

Equipment for particle size analysis:

• Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA )

• Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA )

• Limiting orifice 3 L/min (Millipore Corporation, Biller ica, MA 01821, USA )

• Samp ling probe internal diameter 7 mm

• Balance Sartorius M3P

- 000V001 and LC 1201S

- 00MS (Sa r torius AG, Göttingen, Germany)

Sampling for particle size analyses:

Preweighed m etal collecting discs and a backup particle filter were placed in to the cascade impactor: O ne sample was taken in each concentration per week at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.

Table 11 Sample numbers and volumes for particle size measurement by cascade impactor
Test group Number of samples Sample Volume (L)
1 15 720
2 15 150
3 15 30

Method of analysis: Gr avimetrical determination analyses

The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting and backup filter before and after sampling.

The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter.

Evaluation:

The calculation of the particle size distribution was carried out on the basis of mathematical methods for evaluating particle measurements

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### 3.6.3.3. Particle size analysis by Aerodynamic Particle Sizer (APS)

Particle s ize distribution in the test atmospheres was measured by TSI APS 3321 . Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) is obtained directly by the equipment used (TSI APS 3321).

Frequency:

For each test group on two days during the exposure period, with 3 repeats on each day

3.6.4. Food analyses

The food used in the study was assayed for chemical as well as for microbiological contaminants.

3.6.5. Drinking water analyses

The drinking water is regularly assayed for chemical contaminants by the municipal authorities of as well as for the presence of microorganisms by a contract laboratory.

3.6.6. Bedding and enrichment analyses

The bedding and enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

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### 3.7. EXPERIMENTAL PROCEDU RE

3.7.1. Acclimatization and exposure period

The animals were delivered and subjected immediately to the acclimatization period in which they were adapted to the surroundings.

Prior to the pre

- exposure period, the animals were distributed according to weight a mong the individual test groups. The weight variation of the animals used did not exceed ± 20 percent of the mean weight. The list of randomization instructions was compiled with a computer.

3.7.2. Exposure systems; exposure of the animals

Head

- nose exposure systems

The inhalation atmosphere was maintained inside aerodynamic exposure consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone

- shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.

Two inhalation systems are used. Prior to exposure, to adjust the animals to the exposure condition, INA 120 (V ≈ 155 L, ) was used

. During substance exposure, INA 60 (V ≈ 90 L, ) was used for test groups 1 to 3, and INA 120 for the controls.

Exposures

The head

- nose exposure technique was preferably selected for this dust inhalation study to mini mize fur contamination of the animals with the substance, which cannot be avoided during whole

- body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nom inal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult.

Furthermore, by using the dynamic mode of operation with a low

- volume chamber, the equilibrium characteristic of this exposure technique is favorable: t 99 

### (the time to reach 99% of the final target concentration) is shorter as compared to whole

- body chambers with a higher chamber volume.

A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.

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### In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure ( pre

- exposure period). Then all test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures.

The animals did not have access to water or feed during the exposure.

Figure 2 : Exposure syst em

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### 3.7.3. Measurements of the exposure conditions

Recording of exposure parameters was performed according to the following table.

Table 12 : Recording of exposure parameters
Exposure parameters Determination method Recording Test Group 1 to 3 Supply air (conditioned) Rotameter three times per exposure Supply air (compressed) Rotameter three times per exposure Exhaust air Rotameter three times per exposure Exhaust air out of the mixing tube Rotameter three times per exposure

Chamber humidity Dielectric probe (Testo) three times per exposure Chamber temperature Thermosensor three times per exposure Real time concentration surveillance Scattered light photometers line recorder

Test Group 0 Exposure parameters Determination method Recording Supply air (conditioned) orifice plate with differential pressure measurement automated system

Supply air (compressed) orifice plate with differential pressure measurement automated system

Exhaust air orifice plate with differential pressure measurement automated system

Chamber humidity Dielectric probe (Testo) automated system Chamber temperature Thermosensor automated system

No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

Principles of recording with the automated measuring system:

Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0 - 20 or 4 - 20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.

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### Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data. Relevant disturbances are reported under “ Results”.

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### 3.8. CLINICAL EXAMINATION S

3.8.1. Mortality

The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

3.8.2. Clinical observations

The clinical condition of the test animals was recorded once during the pre

- exposure period and on post

- exposure observation days and at least 3 times (before, during and after exposure) on exposure days.

During exposure only a group wise examination was possible.

3.8.3. Body weight data

The body weight of the animals was determined at the start of the pre

- exposure (day

- 4) , at the start of the exposure period (day 0), on study days 2, 3 (group 3 only), 4, 7, 11, 14, 18, 21, 25 and 28 (test groups 0, 1 and 2).

Body weight change was calculated as the difference between body weight on Monday and Friday of the same week. Group means were derived from the individ ual differences.

Due to clinical signs of toxicity and lethality observed in test group 3, b ody weights were determined additionally on study days 2 and 3 in surviving animals of this group

.

3.8.4. Urine collection

Due to premature death in test group 3, urine samples were collected in 4 surviving animals (No. 50 to 53) from study day 2 to 3, as well as in one concurrent control animal (No. 46). Animal No. 52 died during the night, but still provided a small amount of urine for analysis.

Urine samples were collected in recovery animals in control (animal No. 76 to 80) and test group 2 animals (animal No. 106 to 110) assigned for lavage (Table 6) . After the last exposure and respective clinical observation after the exposure, the animals we re transferred into metabolism cages (with access to water). Urine was collected over night for a total of approximately 18 hours. Urine samples were analyzed for metal ion (Li, Mn, Co, Ni) to determine their urinary excretion

. The whole procedure was repe ated again starting from study day 17 until study day 18. Aliquot s of the urine were examined for creatinine concentration at

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### 3.8.5. Rectal temperature

On st udy day 2, rectal temperature was determined in all surviving animals. For animals exposed on this day, the measurement wa s performed after exposure.

On study day 3, rectal temperature wa s determined in all surviving animals of test group 3.

On study day 4, rectal temperature wa s determined in all surviving animals of test group 3 and in recovery group animals (n = 5) designated for lavage (test group 0, 1 and 2) . For animals exposed on this day, the measurement wa s performed after exposure.

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### 3.9. LUNG BURDEN

Due to premature death of group 3 animals, the lung s of 4 dead animals ( No. 47, 48, 49, 55) were examined for metal ions. Other organs were not preserved. No further animals of test group 3 were examined for organ burden. All samples were st ored at

- 80 °C until analysis.

With exception of test group 3 animals, the animals intended for determination of organ burden (c.f. 3.4) were sacrificed under Narcoren ® 

### (pentobarbital sodium) anesthesia by exanguination from the abdominal aorta and vena cava. The following org an s and tiss u es

were stored at - 80°C.

1. Lung 2. Lymph nodes (mediastinal + hilar) 3. Liver 4. Spleen 5. Kidney 6. Basal brain with olfactory bulb

The following organs and tissues were examined

.

Table 13 List of organs and tissues of respective animals analyzed for metal ions
Sampling day Group 0 Group 1 Group 2 Group 3

Lung day 2 9 * 47

- 49, 55

Lung day 25 84 99

- 101 114

- 116 Liver, kidney, spleen day 25 84 99

- 101 114

- 116 Blood day 2 1

- 5 46, 50

- 53 *animal No. 9 was sacrificed on study day 4, but was analyzed with 47

- 49 and 55 together. It serves as back ground control for the analysis



### The extra

- pulmonary organs liver, kidney, spleen were only examined after 3 weeks recovery period, in order to examine the accumulation potency of the metal ions.

All samples were examined for metal ions (Li, Mn, Co, Ni) at

All other samples were stored in the freezer (

- 80°C) and were not analyzed. Analysis may be performed at a later time point. If no analysis is requested, the samples will be discarded one year after finalization of the report.

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### 3.10. CLINICAL PATHOLOGY

In the morning blood was taken from the retroorbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were

car ried out in a randomized sequence.

The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.

The results of clinical pathology examinations were expressed in International System (SI) units.

The following examinations were carried out:

3.10.1. Hematology

The following parameters were determined in blood with EDTA -K 3 

### as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non

- ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6 th 

### Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

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### Table 14 : Parameters and methods of hematology

Parameter Unit Method References Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH Hematocrit (HCT) L/L calculation: MCV x erythrocytes Mean corpuscular volume (MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram) Operator’s Guide for Advia 120 System

Mean corpuscular hemoglobin (MCH) fmol calculation:

hemoglobin erythrocytes Mean corpuscular hemoglobin concentration (MCHC) mmol/L calculation:

hemoglobin hematocrit Platelet count (PLT) giga/L flow cytometric laserlight scattering Differential blood count % and giga/L cytochemistry coupled with flow cytometry Reticulocytes % cytochemistry coupled with flow cytometry



### Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

Table 15 : Parameter and method of clotting test

Parameter Unit Method References Prothrombin time (Hepato Quick’s test) (HQT) seconds citrated blood with calcium thromboplastin Fischer, M. and Falkensammer, Ch., Klin. Wschr. 86 , 577

- 583 (1974)



### 3.10.2. Clinical chemistry

An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters

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### Table 16 : Parameters and methods of clinical chemistry

Enzyme (systematic name and system number) Unit Method, wave

length and measuring temperature References

Alanine aminotransferase (ALT) (L

- alanine: 2

- oxoglutarate aminotransferase; EC 2.6.1.2.) μkat/L kinetic UV test, 340 nm; 37°C Recommendations of the German Society for Clinical Chemistry: "Standardization of methods for determining enzyme activities in biological liquids". Aspartate aminotransferase (AST) (L

- aspartate: 2

- oxoglutarate aminotransferase; EC 2.6.1.1.) μkat/L kinetic UV test, 340 nm; 37°C J. Clin. Chem. Clin. Biochem. 8 , 658

- 660 (1970); J. Clin. Chem. Clin. Biochem. 9 , 464

- 465 (1971); J. Clin. Chem. Clin. Biochem. 10

, 182

- 192 (1972) Alkaline phosphatase (ALP) ( orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.) μkat/L kinetic color test, 415 nm, 37°C Roche working instructions

γ - Glutamyltransferase (GGT) ( γ

- glutamyl) peptide: aminoacid

- γ glutamyl

- transferase; EC 2.3.2.2.) nkat/L kinetic color test, 415 nm, 37°C Szasz, G. et al., J. Clin. Chem. Clin. Biochem. 12 , 228 (1974) Roche working instructions



### Page 42 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsBlood Chemistry Parameter Unit Method References

Sodium (NA) mmol/L

Potassium (K) mmol/L ion selective electrodes (ISE) Hitachi 917

- working instructions

Chloride (CL) mmol/L

Inorganic phosphate (INP) mmol/L molybdate reaction Henry, R.J. in: "Clinical Chemistry", Harper and Row Publishers, New York (1974); Roche working instructions Calcium (CA) mmol/L o

- cresolphthalein complex without deproteinization Ray Sarkar, B.C. and Chauhan, U.P.S., Anal. Biochem. 20 , 155 (1967); Roche working instructions Urea (UREA) mmol/L enzymatic determination with the urease/ glutamate dehydro

genase method Neumann, U. and Ziegenhorn , J.: XVI, Nordiska kongressen for klinisk kemi och klinisk fysiologi 1977, Oulu, Finland; Roche working instructions Creatinine (CREA) μmol/L kinetic Jaffé method without deproteinization Bartels, H. et al., Clin. Chim. Acta 37 , 193 (1972); Roche working instructions Glucose (GLUC) mmol/L hexokinase/glucose

- 6

- phosphate dehydrogenase method Schmidt, F.H., Klin. Wschr. 39 , 1244

- 1247 (1961); Roche working instructions Total bilirubin (TBIL) μmol/L DPD method Wahlefeld, A.W. et al., Scand. J. Clin . Lab. Invest. 29 , Suppl. 126 (1972) Abstract 11.12; Roche working instructions Total protein (TPROT) g/L biuret method Weichselbaum, T.E., Amer. J. Clin. Path. 10 , 40 (1946); Roche working instructions Albumin (ALB) g/L bromocresol green method Doumas et al., Clin. Chim. Acta 31 , 87 (1971); Roche working instructions Globulins (GLOB) g/L difference between total protein and albumin Triglycerides (TRIG) mmol/L enzymatic color test with lipase esterase/ glycerokinase/ glycerol

3- phosphate oxidase/4

- amino

phenazone mod. method by Wahlefeld, A.W., in "Methoden der enzymatischen Analyse" [Methods of enzymatic analysis] (Bergmeyer, H.U., ed.) Vol. II, 3rd ed., Verlag Chemie Weinheim, GERMANY, pp. 1878

- 1882 (1974); Roche working instructions Cholesterol (CHOL) mmol/L enzymatic determination with cholesterol esterase/ cholesterol oxidase/4

- amino

- phenazone (CHOD

- PAP method) Siedel, J. et al., J. Clin. Chem. Clin. Biochem. 19 , 838 (1981); Roche working instructions

Magnesium (MG) mmol/L xylidylb lue method Mann, C.K. and Yoe, J.H., Anal. Chem. 28

, 202

- 205 (1956) Bohnon, C., Clin. Chim. Acta 7 , 811

- 817 (1962)



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### 3.10.3. Acute phase proteins in serum

Rat α 2- macroglobulin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg , OR, USA (cat. no. E

- 25A2M)

. Rat haptoglobin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E

- 25HPT).

Both ELISA kits were measured with a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer.

3.10.4. Bronchoalveolar lavage fluid (BAL)

The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren ® 

### anesthesia. The lung was lavaged by two instillations of physiologic saline.

The following examinations were carried out in 5 male animals per test group.

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### 3.10.4.1. Cytology in BAL

Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically.

Table 17 : Parameters and methods of cytological parameters in BAL

Parameter Unit Method References Total cell count (BALTCN) cn/μL BAL cytochemistry coupled with flow cytometry Operator’s Guide for Advia 120 System Macrophages (BALMPH) % and cn/μL BAL Stained according to Warheit, D. B. and Hartsky, Polymorphonuclear neutrophils (BALPMN) % and cn/μL BAL Wright; microscopic evaluation M. A. Microsc. Res. Techn. 26 , 412

- 422 (1993) Lymphocytes (BALLY) % and cn/μL BAL Eosinophils (BALEO) % and cn/μL BAL Monocytes (BALMO) % and cn/μL BAL Atypical cells (BALATY) % and cn/μL BAL



### 3.10.4.2. Total Protein and enzymes in BAL

An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the humoral parameters in the bronchoalveolar lavage fluid.

Table 18 : Parameters and methods of total protein and biochemical parameters in BAL

Parameter Unit Method References

γ− Glutamyltransferase (GGT) ( γ - glutamyl) peptide: aminoacid

- 

- glutamyl

transferase; EC 2.3.2.2.) nkat/L Kinetic color test, 415 nm, 37°C Szasz, G. et al., J. Clin. Chem. Clin. Biochem. 12 , 228 (1974) Roche working instructions

Protein (MTP) mg/L Benzethoniumchloride method Iwata, I., Nishikase, O., Clin. Chem. 35/8: 1731

- 1734 (1989) Roche working instructions Lactate dehydrogenase (LDH) (L

- Lactate

- NAD

oxidoreduktase; EC 1.1.1.27) μkat/L Kinetic UV test, 340 nm; 37°C Recommendations of the German Society for Clinical Chemistry: "Standardization of methods for determining enzyme activities in biological liquids". J. Clin. Chem. Clin. Biochem. 8 , 658

- 660 Roche working instructions Alkaline phosphatase (ALP) (ortho

- phosp horic acid monoester phosphohydrolase EC 3.1.3.1.) μkat/L Kinetic UV test, 340 nm; 37°C (1970); J. Clin. Chem. Clin. Biochem. 9 , 464

- 465 (1971); J. Clin. Chem. Clin. Biochem. 10 , 182

- 192 (1972); Roche working instructions
N- acetyl

- β

- Glucosaminidase (NAG) (2

- Acetamido -2- deoxy

- β -D- glucosideacet

amidodeoxy

- glucohydrolase; (EC 3.2.1.30) nkat/L Color test, 580 nm; 37°C Yakata, M. et al., Jap. J. Clin. Path. Suppl. 56 , 90 (1983); Roche working instructions



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### 3.11. PATHOLOGY

The animals were sacrificed under Narcoren ® 

### ( pentobarbital sodium) anesthesia by exsanguination from the abdominal aorta and vena cava . The exsanguinated animals were necropsied and assessed by gross pathology.

3.11.1. Necropsy

Animals that died intercurrently or were sacrificed moribund were necropsied and assessed by gross pathology as soon as possible after their death.

The following tables show extend s of the weight assessment, gross and / or histopathological examination:

Pathology tables are divided into 3 subsets : F1, R1 and R2 , animals are allocated as follows:

Table 19 Subset F1: including all surviving main group animals intended for light microscopy in the study plan

Test group 0 1 2 3 Animal Nos. 6

- 8 21

- 23 36

- 38 No survivors * (51

- 53) Weights X X X

-- Gross pathology X X X Only animal 52

Histopathology X X X

--
*animal 52 was examined macroscopically but not by light microscopy as it was transferred to the blood sampling, lavage group in amendment 1, animals 51 and 53 were transferred to the recovery group R1 in amendment 1 and examined histopathologically after a 2 week recovery period.

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### Table 20 Subset R1: including all surviving recovery group animals intended for light microscopy in the study plan and all surviving animals from group 3 which were originally not intended for pathology (weights and gross pathology) but included for this investigation in amendment 1

Test group 0 1 2 3 Animal Nos. 81

- 83 96

- 98 111

- 113 46,50,51 * ,53 * ,123, 129 Weights X X X X Gross pathology X X X X

Histopathology

-- -- -- Only 51, 53

Table 21 Subset R2: including recovery animals from test group 3 which died before the end of the study (animals 121,125,126,127,128 di ed on study day 2, animal 131 122, 124 died on day 3

Test group 3 121, 122,124,125,126,127,128,131

Weights

-- Gross pathology X

Histopathology X

Not included in the tables were animals 54, 56, and 130, (all test group 3, 50 mg/m3) for those animals findings were noted handwritten on sheets used for acute necropsies which are included in the raw data of this study. For these animals neither organ weight assessment nor histop athology was performed.

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### 3.11.2. Weight parameters

Weight assessment was carried out on all animals listed for this investigation under
necropsy. The following weights were determined:

1. Anesthetized animals 2. Adrenal glands 3. Brain 4. Epididymides 5. Heart 6. Kidneys 7. Liver 8. Lungs 9. Spleen 10. Testes 11. Thymus 12. Thyroid glands

3.11.3. Organ / Tissue preservation list

The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution:

1. All gross lesions 2. Adrenal glands 3. Brain with olfactory bulb 4. Epididymides ( mo dified Davidson 's solution) 5. Heart 6. Kidneys 7. Larynx 8. Liver 9. Lung 10. Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes) 11. Nose (nasal cavity) 12. Pharynx 13. Spleen 14. Testes (modified Davidson 's solution) 15. Thymus 16. Trachea

From the liver, one slice each of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy’s solution and embedded in paraplast.

The liver, testes and epididymides of animals that died or had to be sacrificed intercurrently were fixed in 4% buffered formaldehyde s olution.

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### 3.11.4. Histopathology

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:

Table 22 List of animals and their o rgans and tissues for histopat hology
Organs Test group

0 1 2 3 *
1. All gross lesions A2 A2 A2 A2
2. Nasal cavity (4 levels) d 

### A1 A1 A1 A1
3. Larynx (3 levels) e 

### A1 A1 A1 A1
4. Liver B1 B1 B1 B1
5. Trachea f 

### A1 A1 A1 A1
6. Lungs (5 lobes) A1 A1 A1 A1
7. Lymph nodes (tracheobronchial and mediastinal lymph nodes) A1 A1 A1 A1

* animals to be examined histopathologically are listed under necropsy.

For all animals listed for histopathology under necropsy nasal cavity (4 levels), larynx (3 levels), trachea, lungs (5 lobes) and tracheobronchial and mediastinal lymph nodes were examined.

Methods/Scope of examinations: A = Hematoxylin

- eosin B = Paraplast embedding 1 = All animals/group 2 = All animals affected/group

d One level includes nasopharyngeal duct; the 4 levels allow adequate examination of the squamous, transitional, respiratory and olfactory epithelium, and the draining lymphatic tissue (NALT).

e One level includes the base of the epiglottis.
f One transverse section and one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi.



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### 3.12. STATISTICS

Means, medians and standard deviations of each test group were calculated for several parameters (see tables). Further statistical analyses were performed according to following tables . Details were explained in the summary tables in PART I :

Table 23 : Statistical analyses

Parameter Statistical test markers in the tables References

Body weight, body weight change, rectal temperature A com parison of each group with the control group was performed using DUNNETT's test (two

- sided) for the hypothesis of equal means * for p < 0.05 ** for p < 0.01 DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096 – 1121

DUNNETT, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482 – 491
Blood parameters Fo r parameters with bidirectional changes: Non

- parametric one

- way analysis using KRUSKAL

WALLIS test. If the resulting
p- value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON

- test (t wo

- sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON

- test (one

- sided) for the hypothesis of equal medians * for p < 0.05 ** for p < 0.01 SIEGEL, S. (1956): Non

- parametric statistics for the behavioural sciences. McGraw

- Hill New York

Broncho

- alveolar lavage fluid (BALF) and lung tissue parameters Pairwise comparison of each dose group with the control group using the WILCOXON

- test (one

- sided) for the hypothesis of equal medians * for p < 0.05 ** for p < 0.01 SIEGEL, S. (1956): Non

- parametric statistics for the behavioural sciences. McGraw

- Hill New York



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## 4. RESULTS

Throughout the sections of the report, when intergroup differences are referred to as "significant " it implies that the differences have attained statistical significance (p ≤ 0.05) when compared with the control group.

4.1. ANALYSES

4.1.1. Measurements of atmosphere parameters and operation conditions

Detailed results of the measurements of concentrations and operation conditions can be found in Part II.

Details of the particle size analyses are given in Part II.

Individual values may be found in the record sheets and are kept with the raw data.

4.1.1.1. Concentration measurements in the exposure system

(Tables ID

- 1, IID

- 1 - IID -5)

Table 24 : Study means and standard deviations of test substance concentrations

Test group Target concentration (mg/m3) Measured concentration (mg/m3) N ominal concentration (mg/m3) Mean SD
0

- 1 2 2.2 0. 6 6 .2
2 1 0 10

. 0 1

. 9 22 .0
3 5 0 5 4

. 5 4

. 0 100.3

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### Figure 3 : Time course of daily concentrations

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures. Examples of recorder protocols are depicted in the figure below (original protocols a re archived with the raw data).

Figure 4 : Examples of concentration real time records

Test group 1 ( 21 Sep 2011)

End 6h Start

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### Test group 2 (23 Sep 2011)

End 6h Start

Test group 3 (19 Sep 2011)

End 6 h Start

Real time surveillance of the inhalation atmospheres with scattered light photometers showed some variations of concentration during daily exposure, reflecting the variation found for the study means (examples of recorder protocols see APPENDIX, original p rotocols are archived with the raw data).

Figure 5 : Examples of concentration real time records showing variations

Test group 2 (19 Sep 2011)

End 6h Start

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### 4.1.1.2. Measurements concerning operation conditions

(Tables ID

- 1, IID

- 1 - IID -4)

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.

Daily m ean s of relative humidities in the inhal ation systems ranged between 41 .9 and 59 .9 %. Daily m ean s of temperatures in the inhalation systems ranged between 21. 4 and 22.2 °C.

4.1.1.3. Results of the particle size analyses

(Tables ID

- 1, IID -5 - IID

- 48 )

All measurements of particle size resulted in MMADs between 3.2 and 4.9 μm with GSDs between 2.2 and 3.8. In all cases, t he calculated mass fractions of particles below 3 μm

ae rodynamic size ranged between 31 .5 and 47 .4 %. The generated dust aerosols were generally at the upper range of respirable particles for rats

. The cascade impactor measurement was confirmed by APS.

In t he following table the MMAD/GSD by cascade impactor and APS are presented ( Tables IID -5 - IID

- 10 in Part II):

Table 25 Particle size distributions measured by different devices
Test groups 1 2 3 Target concentration (mg/m 3 ) 2 10 50 MMAD (μm) / GSD 3.8 / 3.8 3. 2 / 2. 6 3

. 4 / 2.2 3.4 / 2. 3 4.9 / 2.8

APS 3321: MMAD (μm) / GSD 3. 23 / 2.16 3. 30 / 2

. 09 3. 58 / 2

. 13 4

. 00 / 2

. 07 3

. 2 7 / 2

. 10 3.0 9 / 2

. 07



### 4.1.2. Food analyses

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 9 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1*10 5 

### /g food. Individual results are to be found in the archives of the

.

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### 4.1.3. Drinking water analyses

On the basis of the analytical findings the drinking water was found to be suitable. served as a guideline for maximum tolerable contaminants. Individual results are to be found in the archives of the

4.1.4. Bedding and enrichment analyses

On the basis of the analytical findings the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov

- Dec 1979, pp. 24

- 33, served as a guideline for maximum tolerable contaminants. Individual results are to be found in the archives of the

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### 4.2. CLINICAL EXAMINATION S

Summary Tables of the results can be found in Part I (Tables IA

- 1 - IA -6 ); individual values are to be found in Part II (Tables IIA

- 1 - IIA

- 15 ).

4.2.1. Mortality

(Summary tables IA

- 1 - IA

- 3 in Part I and individual value tables IIA

- 1 - IIA

- 11 in Part II)

Lethal ity was observed only in test group 3 animals after 2 exposures

. Total 11 animals were found dead on study day 2, further 3 animals were found dead on study day 3 and 2 animals were sacrificed i n a moribund state on study day 3

.

Total 16 of the 22 animal s were found dead or sacrificed i n a moribund state within the first days.

4.2.2. Clinical observations

(Summary tabl es IA

- 1 - IA -3 in Part I and individual value tables IIA

- 1 - IIA

- 11 in Part II)

Clinical signs of toxicity were only observed in test group 3 animals. On study day 2, all surviving animals of group 3 showed accelerated and intermittent respiration. Most of them were in a poor general condition ; pale skin and piloerection were observed

. A few of these animals were found dead on study day 3 or sac rificed i n a moribund state. The clinical signs of toxicity declined in the remaining animals during the next days of exposure free period. No more clinical signs of toxicity were observed from study day 10 onward.

4.2.3. Body weight data

(Summary tables IA -4 - IA -6 in Part I and Individual values tables IIA -12 - IIA -15 in Part II)

Body weight:

The mean body weights of the group 1 and 2 animals were not statistically significantly different from the control group 0 throughout the study

. In test group 3 (50 mg/m3) the mean body weights were significantly lower than the control on study day 2. The mean body weights of the surviving animals were significantly lower on study day 4. This effect was not observed on study day 7 onward.

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### Figure 6 : Body weight development of male animals

Prior to exposure: study day

- 5 to -1 Exposure period: study day 0 to 4 Post exposure period: study day 5 to 28



### Body weight change

The body weight changes of test group 1 and 2 animals were comparable to the controls throughout the study

. The following statistically significant body weight changes were observed in test group 3 animals:

Significantly reduced f rom study day 0 to 4: group 3 Significantly increased f rom study day 14 to 18: group 1 and group 3

The reduced body weight gain in test group 3 from study day 0 to 4 is considered to be treatment

- related, because lethality occurred and severe clinical signs of toxicity were observed

. From study day 14 to 18, the mean body weight change of test group 1 and 3 was higher than the concurrent control group. This effect is considered to be incidental.

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### 4.2.4. Rectal temperature

(Summary tables IA

- 7 in Part I and Individual values tables IIA

- 16 - IIA

- 19 in Part II)

The mean rectal temperature s of group 1 and 2 animals were comparable to the concurrent control group on both measurement days (study day 2 and 4).

On study day 2, the mean value of rectal temperature of the surviving animals was significantly lower than the concurrent control. This effect was not observed on study day s 3 and 4.

Figure 7 : Mean rectal temperature of male anima ls on study day 2 (test group 0, 1 and 2: n =22, test group 3: n =13)

4.2.5. Urinary excretion of metal ions

Urine samples were collected and examined for metal ion s. Li, Co and Ni were detected in urine in group 2 animals after 5 exposures, in group 3 animals after 2 exposures . The amount of Mn was below detection limit of 0.3 μg Mn which is in the range < 0.005 μg/μmol creatinine

. After recovery period, the detected metal ions were in the control range.

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### Table 26 Urinary excretion of metal ions

Animal No. material found in urine μg/μmol creatinine Co Li Ni Control value Range < 0.005 < 0.01 < 0.005
test group 3 (study day 2

- 3)

mean 0.18 0.40 0.08

test group 2 (study day 4

- 5)

mean 0.10 0.13 0.04



### 4.3. ORGAN BURDEN

In the following table, the amounts of metal ions found are presented. Considering the fractions of respective metal ion in the test substance (analysis of the test substance, Part III), a lung burden was calculated to be between 14 μg (based on Li) to 3 0 μg (based on Co or Ni). This amount is considerably lower than the expected one.

Table 27 Lung burden in dead animals after 2 exposures

Test group Animal No Lung burden Co Li Mn Ni
3 47
3 48
3 49
3 55
0 9



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### 4.4. CLINICAL PATHOLOGY

Summary tables of the results are given in the Appendix of PART I; individual values are given in Part B of PART II.

Blood samples were taken in rats of test groups 1 and 2 (2 and 10 mg/m 3 

### ) at study day 7 and after a 3 weeks recovery period at study day 28. In rats of test group 3 (50 mg/m 3 

### ) venipuncture was performed at study day 2 and again after a 3 weeks recovery period at study day 2 5.

Broncho

- alveolar lavage fluid was sampled in rats of test groups 1 and 2 (2 and 10 mg/m 3 

### ), only. Sampling days were study day 7 and 28.

4.4.1. Hematology

(Tables IB 1 – IB 2; IB 19; IB 24 Red blood cell + coagulation parameters) (Tables IB 3 – IB 4; IB 20; IB25White blood cell parameters)

At study day 7, in rats of test groups 1 and 2 (2 and 10 mg/m 3 

### ) red blood cell (RBC) counts, hemoglobin and h aematoc rit values were higher compared to controls.

At study day 7, in rats of test group 1 and 2 (2 and 10 mg/ m 3 

### ) relative reticulocyte counts were lower compared to controls, and in rats of test group 2 prothrombin time (HQT) was prolonged. However, the means of the mentioned parameters were within historical controls (reticulocyte counts : 1.1

- 3.1 %; prothrombin time: 33.2

- 39.6 sec, PART III, Supplement), whereas the reticulocyte count study control mean was above, and the prothrombin time mean of the controls was below the historical control ranges. Moreover, reticulocyte counts were not changed dose

- dependently. Therefore, the mentioned alterations of reticulocyte counts and prothrombin time were regarded as incidental and not treatment

- related.

At study day 7 in rats of test group 1 (2 mg/m 3 

### ) relative neutrophil counts were higher and relative lymphocyte counts were lower compared to controls, without any changes of the absolute cell counts. Both parameters were not changed dose

- dependently and therefore the alterations were regarded as incidental and not treatment

- related.

After the 3

- weeks

- recovery period, in rats of test group 3 (50 mg/m 3 

### ) relative reticulocyte counts and relative basophil counts as well as absolute large unstained cell (LUC) counts were lower compared to controls. All values were within historical control ranges (relative reticulocyte count s 1.1

- 3.1 %; relative basophil counts 0

- 0.9 %, absolute LUC counts 0.01

- 0.05 Giga/L, PART III, Supplement) and therefore, these changes were regarded as incidental and not treatment

- related.

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### 4.4.2. Clinical chemistry

(Tables IB 5 – IB 6; IB 21; IB 26 Enzymes) (Tables IB 7 – IB 8; IB 22; IB 27 Substrates) (Tables IB 9 – IB 10; IB 23; IB 28 Electrolytes + minerals)

At study day 7, in rats of test group 2 (10 mg/m 3 

### ) alkaline phosphatase activities were increased.

At study day 2, in rats of test group 3 (50 mg/m 3 

### ) sodium levels were higher, but inorganic phosphate levels were lower compared to controls. The means of both parameters were within historical control ranges (sodium: 139.1

- 146.0 mmol/L; inorganic phosphate: 1.73

- 2.39 mmol/L, PART III, Supplement) and therefore these alterations were regarded as incidental and not treatment

- related.

After the recovery period at study day 25, in rats of test group 3 (50 mg/m 3 

### ) γ - glutamyl transferase (GGT) were lower compared to controls. This alteration cannot be correlated to any patho

- physiological finding and therefore it was regarded as incidental and not treatment

related.

4.4.3. Acute phase proteins

(Tables IB 11 – IB 12)

No treatment

- related changes of acute phase protein levels were measured after the administration as well as after the recovery period.

4.4.4. Bronchoalveolar lavage fluid (BAL)

4.4.4.1. Cytology in BAL

(Tables IB 13 – IB 16 BAL: cytology )

Table 28 : Changes in mean absolute cell counts in BAL (x

- fold of concurrent control) on study day 7 (2 days after last exposure) and study day 28 (3 weeks after last exposure).

Analyte Study day 7 Study day 28 Gr. 1 2 mg/m 3 Gr. 2 10 mg/m 3 Gr. 1 2 mg/m 3 Gr. 2 10 mg/m 3
Total Cells 4.6** 3.9** 1.3 1.7 Eosinophils 13.4* 22.9** 0.6 0.0 Lymphocytes 54.8** 28.7** 1.4 3.9 Macrophages 1.7* 1.7 1.3 1.7 Neutrophils 219.7** 156.9** 0.7 1.7 Monocytes +** +** 0.0 2.6 Atypical cells +* +* 0.0 3.9 Two

- sided Wilcoxon

- test: * : p ≤ 0.05; ** : p ≤ 0.01 + increase could not be calculated because of zero activity in controls



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### At study day 7, in broncho

- alveolar lavage fluid (BALF) of rats in test group 1 and 2 (2 and 10 mg/m 3 

### ) total cell counts were increased, although not dose

- dependently. This was mainly due to an increase of absolute and relative polymorphonuclear neutrophil (PMN) and lymphocyte counts. Absolute and relative eosinophil, monocyte and atypical cell counts were moderately increased.

Ab solute macrophage counts were marginally higher compared to controls (not statistically significant in test group 2), resulting in a decrease of relative macrophage counts. The changes of the macrophage counts were regarded as not adverse.

After a 3

- weeks

- recovery period, cell counts in BALF of rats of both dose groups were not anymore different from those of controls.

4.4.4.2. Total Protein and enzymes in BAL

(Tables IB 17 – IB 18; BAL: enzymes + protein)

Table 29 : Changes in mean total protein and enzyme levels in BAL (x

- fold of concurrent control) on study day 7 (2 days after last exposure) and study day 28 (3 weeks after last exposure)

Analyte Study day 7 Study day 28 Gr. 1 2 mg/m 3 Gr. 2 10 mg/m 3 Gr. 1 2 mg/m 3 Gr. 2 10 mg/m 3
Total Protein 7.9** 14.6** 1.0 1.4 GGT 8.6** 25.2** 1.0 1.9* LDH 14.7** 22.6** 0.9 1.4 ALP 2.3** 4.6** 0.5 0.5 NAG 2.9** 9.6** 0.8 1.3 GGT = γ - Glutamyl

- transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = β -N- Acetyl glucosaminidase , Two

- sided Wilcoxon

- test: * : p ≤ 0.05; ** : p ≤ 0.01



### At study day 7, in BALF of rats in test groups 1 and 2 (2 and 10 mg/m 3 

### ) total protein, γ glutamyl transferase (GGT), and lactate dehydrogenase (LDH) activities were moderately increased. Alkaline phos phatase (ALP) and β -N- acetyl glucosaminidase (NAG) activities were marginally increased in rats of test group 1 (2 mg/m 3 

### ) and moderately increased in those of test group 2 (10 mg/m 3 

### ).

After the 3

- weeks

- recovery period, all mentioned parameter values were within the normal range. GGT activities in rats of test group 2 (10 mg/m 3 

### ) were statistically increased, but the mean was only slightly above the border of the historical control range (GGT: 0

- 41 nkat/L, PART III, Supplement) and therefore this alteration was regarded as not treatment

- related.

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### 4.5. PATHOLOGY

Summary tables of the results are to be found in Part C of PART I; individual tables can be found in Part C of PART II.

4.5.1. Weight parameters

Main Group (F1) (Tables IC 1 - 4)

Absolute weights When compared to control group 0 (set to 100%), the mean absolute weights of the lungs were increased in a dose - responsive way

. Table 30 Relative increase of absolute organ weight

Male animals Test group (mg/m3) 1 ( 2 ) 2 ( 10 ) 3 ( 50 ) Lungs 111 % 127% N/A (no survivors) 

### Statistics were not calculated as there were only 3 animals per group

Relative weights When compared to control group 0 (set to 100%), the mean relative weights of the lungs were increased in a dose - responsive way.

Table 31 Relative increase of relative organ weight

Male animals Test group (mg/m3) 1 ( 2 ) 2 ( 10 ) 3 ( 50 ) Lungs 101 % 129 % N/A (no survivors) 

### Statistics were not calculated as there were only 3 animals per group

Recovery group (R1) (Tables IC 5 - 8)

Absolute and relative weights None of the weight parameters in the recovery group R1 showed a clear dose response.

Premature decedents (R2) No weights were taken for animals in recovery group R2 as all of those animals were prematurely dead.

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### 4.5.2. Gross lesions (Tables IC 9 - 11 and raw data sheets)

Main Group (F1) test group 3 (50 mg/m3) showed a red discoloration of all lung lobes. of test group 2 showed an enlarged mediastinal lymph node.

Recovery Group (R1) No abnormalities were detected.

Premature decedents (R2) All animals showed a red discoloration of all lung lobes accompanied in one animal each by thoracic effusion or edema.

Not included in the tables were animals 54, 56, and 130, for those animals macroscopic findings only were noted handwritten on sheets used for acute necropsies which are included in the raw data of this study.

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### 4.5.3. Histopathology (Tables IC 12 - 18)

F1 : Main group animals

Animal 52 of test group 3 (50 mg/m3) was examined macroscopically but not by light microscopy as it was transferred to the blood sampling, lavage g roup in amendment 1.

Lungs Bronchial epithelial degeneration and regeneration was noted in all treated animals, peribronchial inflammation was seen in all test group 2 (10 mg/m3) animals.

Table 32 Histological findings in lung s Lungs Male animals Test group (mg/m3) 0 ( 0 ) 1 ( 2 ) 2 ( 10 ) 3 ( 50 ) No. of animals 3 3 3 0 Degeneration /regeneration bronchial epithelium 3 3

• Grade 1 1

• Grade 2 2 3 Inflammation, peribronchial 3

• Grade 2 3



### Mediastinal lymph node In the mediastinal lymph node, lympho

- reticulocellular hyperplasia was observed in all treated animals of test group 1 (2 mg/m3) and 2 (10 mg/m3).

Table 33 Histological findings in mediastinal lymph node
Mediastinal lymph node Male animals Test group (mg/m3) 0 (0) 1 (2) 2 (10) 3 (50) No. of animals 3 3 3 0 Hyperplasia, lympho

- reticulocellular 3 3

• Grade 1 3 2

• Grade 2 1



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### Nasal cavity, level IV Necrosis of the olfactory epithelium was noted in animals of test group 1 (2 mg/m3) and 2 (10 mg/m3).

Table 34 Histological findings in nasal cavity
Nasal cavity, level IV Male animals Test group (mg/m3) 0 (0) 1 (2) 2 (10) 3 (50) No. of animals 3 3 3 0 Necrosis, olfactory epithelium 2 3

• Grade 1 2 3



### Trachea Degeneration/regeneration of the respiratory epithelium was seen in the trachea in animals of test group 1 (2 mg/m3) and 2 (10 mg/m3).

Table 35 Histological findings in trachea

Trachea Male animals Test group (mg/m3) 0 (0) 1 (2) 2 (10) 3 (50) No. of animals 3 3 3 0 Degeneration/regeneration, epithelium 3 3

• Grade 1 3 3



### Tracheobronchial lymph node In the tracheobronchial lymph node, lympho

- reticulocellular hyperplasia was observed in all treated animals of test group 1 (2 mg/m3) and 2 (10 mg/m3).

Table 36 Histological findings in tracheobronchial lymph node
Tracheobronchial lymph node Male animals Test group (mg/m3) 0 (0) 1 (2) 2 (10) 3 (50) No. of animals 3 3 3 0 Hyperplasia, lympho

- reticulocellular 2 3

• Grade 1 2

• Grade 2 3



### R1: Recovery animals The two animals of test group 3 (50 mg/m3) showed minimal degeneration and regeneration of the bronchial epithelium after 2 weeks recovery period, while none of the other organs examined showed treatment – related findings.

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### R2: Premature decedents

Incidences and grading of all microscopic findings of group R2 are listed in the following tables, not all organs could be examined for all animals as some were autolytic and not possible to evaluate.

Some findings varied with time after first exposure, a comparison is presented below the following incidence tables.

Larynx, level I and II Squamous metaplasia was seen in the larynx with incidence and grading according to the tables below:

Table 37 Histological findings in larynx (level I and II) of premature decedents

Male animals

Test group (mg/m3) 3 ( 50 ) Larynx, level I No. of animals 8 Squamous metaplasia 2

• Grade 1 1

• Grade 2 1 Larynx, level II Squamous metaplasia 8

• Grade 1 4

• Grade 2 3

• Grade 3 1



### Lungs The following findings were present in the lungs, they varied with survival time as described later

. Alveolar fibrin formed hyaline membranes over alveolar septae. Inflammation consisted of varying amounts of neutrophils, macrophages and eosinophils, sometimes eosinophil granules were free in alveolar spaces. Degeneration/regeneration of the bronchial epi thelium was characterized by sloughed epithelial cells (degeneration) and mitotic figures in the epithelium (regeneration).Diffuse hyperemia correlated with reddened lungs at necropsy.

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### Table 38 Histological findings in lung s of premature decedents
Lungs Male animals Test group (mg/m3) 3 ( 50 ) No. of animals 8 Fibrin, alveolar 6

• Grade 2 3

• Grade 3 3 Inflammation, multifocal 8

• Grade 2 3

• Grade 3 3

• Grade 4 2 Hyperemia, diffuse 8

• Grade 2 2

• Grade 3 6 Type II pneumocyte proliferation 3

• Grade 3 1

• Grade 4 2 Eosinophil degranulation 4

• Grade 1 2

• Grade 2 2 Degeneration/ regeneration bronchial epithelium 4

• Grade 2 4



### Mediastinal lymph node In the mediastinal lymph node, lympho

- reticulocellular hyperplasia was observed with incidence and grading according to the tables below:

Table 39 Histological findings in mediastinal lymph nodes of premature decedents
Mediastinal lymph node Male animals Test group (mg/m3) 3 ( 50 ) No. of animals 6 Hyperplasia lympho

- reticulocellular 6

• Grade 2 4

• Grade 3 2



### Nasal cavity Degeneration of the olfactory epithelium was noted in some animals in level III of the nasal cavity, while necrosis of the olfactory epithelium and squamous metaplasia of the olfactory epithelium were seen in level IV of the nasal cavity. Some findings in the nasal cavity varied with survival time as described later. Incidence and grading of findings in the nasal cavity are presented below:

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### Table 40 Histological findings in nasal cavity of premature decedents

Male animals Test group (mg/m3) 3 ( 50 ) No. of animals 5 Nasal cavity, level III Degeneration, olfactory epithelium 2

• Grade 1 2 Nasal cavity, level IV Necrosis olfactory epithelium 5

• Grade 1 4

• Grade 2 1 Squamous metaplasia, olfactory epithelium 4

• Grade 1 4



### Trachea

Multifocal , minimal inflammation was seen in all animals, characterized by a lymphocytic infiltrate and degeneration/ regeneration of the epithelium was seen in one animal.

Table 41 Histological findings in trachea of premature decedents

Trachea Male animals Test group (mg/m3) 3 ( 50 ) No. of animals 8 Inflammation, multifocal 8

• Grade 1 8 Degeneratio/ regeneration, epithelial 1

• Grade 1 1



### Tracheobronchial lymph node In the tracheobronchial lymph node, lympho

- reticulocellular hyperplasia was observed with incidence and grading according to the tables below:

Table 42 Histological findings in tracheobronchial lymph node of premature decedents
Tracheobronchial lymph node Male animals Test group (mg/m3) 3 ( 50 ) No. of animals 7 Hyperplasia lympho

reticulocellular 7

• Grade 1 1

• Grade 2 5

• Grade 3 1



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### Comparison of findings varying with survival time died on day 2 (directly after 2nd exposure) and showed diffuse peracute alveolar damage with alveolar septal hyperemia and alveolar hyaline membranes (fibrin) and also eosinophil degranulation in the lungs. died on day 3 (later after 2nd exposure). Hyperemia, alveolar hyaline membranes, beginning type II pneumocyte proliferation, inflammation and also eosinophil degranulation was seen in the lungs. In the nasal cavity, level IV, there was squamous metaplasia of the olfactory epithelium. died on day 3 (later after 2nd exposure).Type II pneumocyte proliferation and inflammation were noted in the lungs . In the nasal cavity, level IV, there was squamous metaplasia of the olfactory epithelium.

Other findin gs did not show a relation to time.

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## 5. DISCUSSION

During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust aerosol generation techniques in the concentration range tested. Particle size distribution measurement demonstrated that the aeroso ls were respirable for rats.

Inhalation exposure of 50 mg/m 3 

### Cobalt Lithium Manganese Nickel oxide caused lethality after only two exposures. Upon which , the exposure was terminated in this group . The surviving animals showed transient body weight loss, labored and intermittent respiration

, as well as several unspecific clinical signs of toxicity comprising pale skin, piloerection, poor general condition and hypo the r mia

. The surviving animals recovered within a few days after termination of the exposure. The body weight returned to the control level already on study day 7. O n study day 10 all surviving animals were free of any clinical signs of toxicity. At 2 and 10 mg/m3, no body weight impairment, no clinical signs of toxicity were observed throughout the whole exposure period and the whole recovery period.

In urine Ni, Li and Co was found significantly above the back ground excretion of these metals, as showed by analysis of control urine. This fact indicates that the test substance

was ( partially ) systemically available. After 3

- week recovery period, urinary excretion of the metals was at the control level. In blood, no increased metal content could be detected, probably due to the sensitivity of the method. In the lung the deposition after two exp osures was considerably lower than expected, indicating fast clearance of the substance from the lung. After 3

- week recovery period, lung burden of 2 of the 3 test group 2 animals was marginally higher than the control level, in 1 animal it was below detec tion limit . Extra

pulmonary organ s were only examine d after 3

- week recovery period. In liver, kidney and spleen the levels of Ni, Li, Co and Mn were at the control level or below the detection limit of 0.3 μg per organ. Overall, the analyses of urine, orga n burden were consistent and showed that the test substance was bioavailable and was cleared from the lung.

Lung burden was determined in animals that died on study day 2. The total deposition was found significantly lower than expected, indicating a fast clearance o f the test substance from the lungs. Consistent to this finding, urinary excretion of Li, Ni and Co were significantly above the background level. This finding indicate dissolution process in the lung and the metal ions Li + 

### , Ni + 

### and Co 2+ 

### were systemically available. To examine, whether these ions accumulate in the extra

- pulmonary organs and tissues liver, kidney, spleen were examined for these metal ions. None of them were above the level of the concurrent controls. There was no indicatio n for accumulation.

Regarding clinical pathology, after administration of the compound for 5 days in rats of test group 1 and 2 (2 and 10 mg/m 3 

### ) a hemo

- concentration took place, indicated by higher red blood cell counts, h aematoc rit and hemoglobin values. The reason of a higher alkaline phosphatase activity in rats of the higher test group cannot be elucidated, but a treatment

related effect cannot be excluded.

Regarding local findings in the lungs, an acute inflammation was found in rats of both mentioned test groups, by increase of absolute counts of almost all cell fraction in BALF

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### (except macrophage counts), above all neutrophils, but also lymphocytes and eosinophils

. Total protein levels and all measured enzyme activities in BALF (ALP, GGT, LD H and NAG) were also increased in both test groups. All mentioned changes were no more present after three weeks recovery period

.
Regarding pathology, target organs were larynx, lungs, nasal cavity, trachea, and tracheobronchial and mediastinal lymph nodes.

Premature decedents (test group 3 (50mg/m3) after 2 or 3 days on study)
Minimal to moderate squamous metaplasia was seen in the larynx, level I and II , which was regarded as adverse.

In the lungs , findings varied with survival time from acute lesi ons (alveolar fibrin forming hyaline membranes over alveolar septae, eosinophil degranulation) to more chronic (type II pneumocyte proliferation). Multifocal inflammation of the lung parenchyma and degeneration/ regeneration of the bronchial epithelium wer e observed in all animals. In the nasal cavity , d egeneration of the olfactory epithelium was noted in some animals in level III of the nasal cavity, while necrosis of the olfactory epithelium and squamous metaplasia of the olfactory epithelium were seen in level IV of the nasal cavity, squamous metaplasia was seen only in animals dying after 3 days. Multifocal , minimal inflammation was seen in in the trachea of all animals

. In the mediastinal and tracheobronchial lymph node , lympho

- reticulocellular hyperplasia was observed and regarded to be adaptive and secondary to the inflammation in the lungs. Recovery animals (test group 3 (50mg/m3) after recovery period) The two animals of test group 3 (50 mg/m3) showed minimal degeneration and regeneration

o f the bronchial epithelium of the lungs after a 3 weeks recovery period, while none of the other organs examined showed treatment – related findings, indicating recovery from all findings.

Main group animals (test group 1 (2mg/m3) and 2 (10mg/m3) after 5 days) Minimal to slight bronchial epithelial degeneration and regeneration was noted in the lungs in all treated animals, peribronchial inflammation was seen in all test group 2 (10 mg/m3) animals, which was regarded to be adverse. Necrosis of the olfactory epithelium of the nasal cavity, level IV was noted in 2/3 animals of test group 1 (2 mg/m3) and all animals of test group 2 (10 mg/m3) and was regarded as adverse. Degeneration/regeneration of the respiratiory epithelium was seen in the trachea in all animals of test group 1 (2 mg/m3) and 2 (10 mg/m3) and was regarded as adverse.

In the mediastinal and the tracheobronchial lymph node , lympho

- reticulocellular hyperplasia was observed in almost all treated animals of test group 1 (2 mg/m3) and 2 ( 10 mg/m3) indicating an adaptive immune – response secondary to the inflammation in the lungs.

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## 6. CONCLUSION

Inhalation exposure of 50 mg/m 3 

### Cobalt Lithium Manganese Nickel oxide caused lethality after only two exposures. Upon which , t he exposure was terminated in this group . The surviving animals showed transient body weight loss, labored and intermittent respiration as well as several unspecific clinical signs of toxicity. Histological examination of the premature decedents showed lesions throughout the respiratory tract

. Apart from minimal degeneration and regeneration of the bronchial epithelia, no other morphological changes were observed in surviving animals of test group 3 sacrificed on study day 25

. At 2 and 10 mg/m3 clearly treatment

- related ch anges were observed in lavage fluid, hematology and in histology of the respiratory tract

. The effects in hematology and lavage fluid were fully reversible within 3 weeks recovery period.

Due to findings in hematology, lavage fluid and histology of the respiratory tract, A No Observed Adverse Effect Concentration (NOAEC) could not be established under the current study condition.



## 7. REFERENCES

Kaufmann W, Bader R, Ernst H, Harada T, Hardisty J, Kittel B, Kolling A, Pino M, Renne R, Rittinghausen S, Schulte A, Wöhrmann T, Rosenbruch M. 1 st 

### international ESTP expert workshop: "Larynx squamous metaplasia". A re

- consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment. Exp Toxicol Pathol. 200 9

. Nov;61(6):591

- 603.

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## 8. LIST OF ABBREVIATION S

The following list contains abbreviations and definitions generally used in reports for this study type. This report will not necessarily use all expressions listed below.

8.1. CLINICAL EXAMINATION S

AE = after exposure

a.m. = ante meridiem (before noon)

anm = animal BE = before exposure BW = body weight

cm = centimeter d = day D = Dunett ́s test , two sided DE = during exposure f / F = female food cons. = food consumption food eff = food efficiency FST = foot

- splay test
g = gram GS F = grip strength forelimbs GS H = grip strength hindlimbs h = hour Interr. = interrupts Interv. = intervall m / M = male mg/m3; mg/m**3 = milligram per cubicmeter

mm = millimeter N = number of animals or samples

n = Student’s test (two

- sided) NC = not calculable NOAEC = No observed adverse effect concentration
p = significance level
p.m. = post meridiem (after noon) REAR = rearing SPF = specific pathogen free SD = standard deviation %dev = per cent deviation % = per cent

- = not detected

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### 8.2. ANALYTICAL DATA AND EXPOSURE PARAMETERS

b = intercept from linear regression d = diameter EACD = effective aerodynamic cutoff diameter GSD = Geometrical standard deviation h = hour INA = H ead

- nose inhalation system IKA = Whole

- body inhalation system L = liter L/min = liter per minute L/h = liter per hour
ppm = parts per million

m = slope of linear regression line (lnEACD versus probit of cumulative mass distribution) m/s = meter per second mL = milliliter mL/h = milliliter per hour m3 /h = cubicmeter per hour
mg = milligram mg/L = milligram per liter mg/h = milligram per hour mg/m 3 

### = milligram per cubicmeter
μm = micrometer MMAD = mass median aerodynamic diameter 50% Pa = Pascal r2 = squared correlation coefficient of regression RF = backup particle filter (for german term “Restfilter”) SD = standard deviation V = volume v/v = volume per volume % = per cent °C = degree Celsius %RH = per cent relative humidity

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### 8.3. CLINICAL PATHOLOGY

SD = standard deviation N = number of values

HEMATOLOGY:

WBC = white blood cells (leukocytes) RBC = red blood cells (erythrocytes) HGB = hemoglobin HCT = hematocrit MCV = mean corpuscular volume MCH = mean corpuscular hemoglobin MCHC = mean corpuscular hemoglobin concentration PLT = platelets RETI = reticulocytes NEUT = polymorphonuclear neutrophils LYMPH = lymphocytes MONO = monocytes EOS = eosinophils BASO = basophils LUC = large unstained cells NEUTA = polymorphonuclear neutrophils (absolute) LYMPHA = lymphocytes (absolute) MONOA = monocytes (absolute) EOSA = eosinophils (absolute) BASOA = basophils (absolute) LUCA = large unstained cells (absolute) ANISO = anisocytosis MICRO = microcytosis MACRO = macrocytosis VAR = anisochromasia HYPO = hypochromasia HYPER = hyperchromasia HQT = prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY:

ALT = alanine aminotransferase AST = aspartate aminotransferase ALP = alkaline phosphatase SGGT = serum

- γ - glutamyltransferase NA = sodium K = potassium

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### CL = chloride INP = inorganic phosphate CA = calcium UREA = urea CREA = creatinine GLUC = glucose TBIL = total bilirubin TPROT = total protein ALB = albumin GLOB = globulins TRIG = triglycerides CHOL = cholesterol MG = magnesium TBA = bile acids

ACUTE PHASE PROTEINS:

HAPT = haptoglobinr A2M = α 2

- macroglobulin

BRONCHOALVEOLAR LAVAGE:

BALTCN = total cell number BALMPH = macrophages BALLY = lymphocytes BALPMN = polymorphonuclear neutrophils BALEO = eosinophils BALMO = monocytes BALATY = atypical cells GGT = γ - glutamyltransferase MTP = total protein LDH = lactate dehydrogenase ALP = alkaline phosphatase NAG = N

- acetyl

- β

- D

- glucosaminidase

CYTOKINES IN BAL

MCP

- 1 = monocyte chemoattractant protein

- 1 IL

- 8 = Cytokine

- induced neutrophil chemoattractant

- 1 M

- CSF = Macrophage colony stimulating factor OSP = Osteopontin

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### CYTOKINES IN LUNG TISSUE

IL

- 1α = interleukin 1α TNF α = tumor necrosis factor

- alpha

UNITS:

mmol/L = millimole/liter μmol/L = micromole/liter nmol/L = nanomole/liter ng/mL = nanogram/milliliter pg/mL = pictogram/milliliter g/L, G/L = gram/liter L/L = liter/liter % = per cent fmol = femtomole = 10 - 15 

### mole fL = femtoliter = 10 - 15 

### liter tera/L = tera/liter = 10 12 

### /liter giga/L = giga/liter = 10 9 

### /liter μkat/L = microkatal/liter nkat/L = nanokatal/liter PPM = parts per million mg/kg = mg/kilogram μL = microliter ML = mL(milliliter)

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### 8.4. PATHOLOGY

KEY TO THE ABBREVIATIONS USED IN TABLES IC and IIC

F1 = final sacrifice groups

g = weight determination in grams

M = male animals (under sex); mean value (on weight level)

mg = weight determination in milligrams

μg = microgram

n = number of values measured for the determination of mean value and standard deviation

SD = standard deviation

% = percentage related to the reference weight in relative organ weight calculations

% dev = deviation in organ or body weight in % when compared to control

Codes used at finding level:

The codes are used for a grading system which takes into consideration either the severity or the number or the size of a microscopic finding.

severity number size grade 1 minimal very few very small grade 2 slight, mild few small grade 3 moderate moderate number; several moderate size grade 4 marked; severe many large grade 5 massive; extreme extensive number extensive size



### Whenever a grading was not used, the microscopic finding was indicated to be present (P).

Page 79 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsIA- 1StudyToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary - Clinical ObservationSex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3day -4 -> -3Animals examinedN22222222normalNADN 22 22 22 22%100.0100.0100.0100.0



### Page 80 of 269

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsIA- 2StudyToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary - Clinical ObservationSex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3day 0 [BE] -> day 4 [AE]Animals examinedN22222222deadN6 6 6 16%27.327.327.372.7found deadN0 0 0 14%0.00.00.063.6sacrificed scheduledN6 6 6 0%27.327.327.30.0sacrificed moribundN0 0 0 2%0.00.00.09.1normalNADN 22 22 22 22% 100.0100.0100.0100.0



### Page 81 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsIA- 3StudyToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary - Clinical ObservationSex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3day 7 -> 28Animals examinedN1616166Animals with signsN0 0 0 5% 0.0 0.0 0.0 83.3deadN 16 16 16 6% 100.0 100.0 100.0 100.0found deadN0 0 0 0% 0.0 0.0 0.0 0.0sacrificed scheduledN 16 16 16 6% 100.0 100.0 100.0 100.0sacrificed moribundN0 0 0 0% 0.0 0.0 0.0 0.0respirationN0 0 0 5% 0.0 0.0 0.0 83.3acceleratedN0 0 0 5% 0.0 0.0 0.0 83.3intermittentN0 0 0 2% 0.0 0.0 0.0 33.3furpiloerectionN0 0 0 1% 0.0 0.0 0.0 16.7normalNADN 16 16 16 6% 100.0 100.0 100.0 100.0



### Page 82 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statisticsn=DUNNETTIA-4StudyToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary Body Weights - BW / Body Weights [g]Sex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3day -4MeanS.d.NDeviation Vs Control248.09.522n246.19.322-0.8247.19.022-0.4247.712.322-0.2day 0MeanS.d.NDeviation Vs Control257.99.022n258.811.8220.3257.812.522-0.1258.016.2220.0day 2MeanS.d.NDeviation Vs Control255.110.822n257.112.6220.8255.813.9220.3230.414.522-9.7**day 3MeanS.d.NDeviation Vs Control0X0X0X220.814.411Xday 4MeanS.d.NDeviation Vs Control256.911.022n260.213.1221.3254.414.822-1.0238.516.26-7.2*day 7MeanS.d.NDeviation Vs Control262.014.316n261.115.016-0.3256.518.116-2.1255.517.46-2.5day 11MeanS.d.NDeviation Vs Control283.99.711n281.716.611-0.7275.018.511-3.1273.118.76-3.8day 14MeanS.d.NDeviation Vs Control297.99.311n294.818.411-1.0290.918.111-2.4287.020.36-3.6day 18MeanS.d.NDeviation Vs Control300.120.411n312.921.1114.3294.223.711-2.0304.921.361.6



### Page 83 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statisticsn=DUNNETTIA-5StudyToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary Body Weights - BW / Body Weights [g]Sex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3day 21MeanS.d.NDeviation Vs Control321.311.111n324.323.5110.9313.420.211-2.4314.222.26-2.2day 25MeanS.d.NDeviation Vs Control319.016.111n325.530.2112.1316.521.511-0.8303.721.76-4.8day 28MeanS.d.NDeviation Vs Control329.14.95n335.921.552.0322.320.95-2.10



### Page 84 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statisticsd = day; n=DUNNETTIA- 6StudyToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary Changes Body Weights - BW / Body Weights [g]Sex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3d -4 -> 0MeanS.d.NDeviation Vs Control9.93.422n12.74.22227.910.76.5227.810.34.9224.5d 0 -> 4MeanS.d.NDeviation Vs Control-1.15.122n1.44.722-226.8-3.45.122213.4-12.38.261,029.1**d 7 -> 11MeanS.d.NDeviation Vs Control14.73.311n15.54.6115.713.75.211-6.917.63.6619.5d 14 -> 18MeanS.d.NDeviation Vs Control2.315.011n18.13.511698.4**3.313.41145.617.93.36687.6*d 21 -> 25MeanS.d.NDeviation Vs Control-2.317.511n1.313.711-155.83.112.711-235.1-10.52.56359.4



### Page 85 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statisticsn=DUNNETTIA - 7Study30-Apr-2013 11:51ToxData© System 3.0Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposureSummary Rectal Temperature (°C)Sex:Male- Phase:In-lifeG 0 / MTest Group 0G 1 / MTest Group 1G 2 / MTest Group 2G 3 / MTest Group 3day 2MeanS.d.N37.60.522n37.30.72237.40.52235.51.713**day 3MeanS.d.N0X0X0X37.31.88Xday 4MeanS.d.N37.30.45n37.90.5536.90.3536.70.46



### Page 86 of 269

This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 1
Study 21-Dec-2011 12:21
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Red blood cell + coagulation parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
RBC [tera/L] day 7 Mean S.d. N Median 7.55 0.41 5 7.54 x 8.27 0.44 5 8.15 * 8.63 0.41 5 8.53 **

HGB [mmol/L] day 7 Mean S.d. N Median 8.3 0.3

5 8.4 x 9.2 0.3

5 9.3 ** 9.4 0.5

5 9.3 **

HCT [L/L] day 7 Mean S.d. N Median 0.409 0.015 5 0.411 x 0.440 0.013 5 0.437 ** 0.453 0.024 5 0.454 *

MCV [fL] day 7 Mean S.d. N Median 54.3 1.4

5 54.5 x 53.3 1.3

5 53.6 52.5 1.5

5 52.2
MCH [fmol] day 7 Mean S.d. N Median 1.11 0.03 5 1.11 x 1.11 0.05 5 1.14 1.09 0.02 5 1.08
MCHC [mmol/L] day 7 Mean S.d. N Median 20.39 0.36 5 20.41 x 20.83 0.51 5 20.99 20.73 0.20 5 20.65
RET [%] day 7 Mean S.d. N Median 6.1 1.1

5 6.2 x 1.8 0.5

5 1.6 ** 2.4 0.7

5 2.3 **

PLT [giga/L] day 7 Mean S.d. N Median 1,039 210

5 1,074 x 889 20

5 886 903 136

5 951
HQT [sec] day 7 Mean S.d. N Median 32.9 0.9

5 32.9 x 34.4 1.5

5 35.3 35.1 1.5

5 34.1 *



### Page 87 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 2
Study 19-Dec-2011 13:57
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Red blood cell + coagulation parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
RBC [tera/L] day 28 Mean S.d. N Median 8.25 0.28 5 8.30 x 8.50 0.38 5 8.61 8.51 0.34 5 8.52
HGB [mmol/L] day 28 Mean S.d. N Median 8.7 0.3

5 8.6 x 9.0 0.2

5 9.0 8.7 0.2

5 8.8
HCT [L/L] day 28 Mean S.d. N Median 0.424 0.010 5 0.420 x 0.437 0.012 5 0.441 0.428 0.011 5 0.426
MCV [fL] day 28 Mean S.d. N Median 51.4 2.3

5 52.7 x 51.4 1.5

5 51.6 50.3 1.0

5 50.6
MCH [fmol] day 28 Mean S.d. N Median 1.05 0.06 5 1.08 x 1.06 0.04 5 1.07 1.03 0.04 5 1.03
MCHC [mmol/L] day 28 Mean S.d. N Median 20.52 0.28 5 20.50 x 20.62 0.19 5 20.59 20.40 0.42 5 20.35
RET [%] day 28 Mean S.d. N Median 2.6 0.5

5 2.4 x 2.4 0.3

5 2.4 2.7 0.4

5 2.9
PLT [giga/L] day 28 Mean S.d. N Median 875 35

5 881 x 910 85

5 920 829 129

5 820
HQT [sec] day 28 Mean S.d. N Median 35.3 1.8

5 35.4 x 35.9 2.0

5 35.2 35.4 1.3

5 35.4



### Page 88 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 3
Study 19-Dec-2011 13:59
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure White blood cell parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
WBC [giga/L] day 7 Mean S.d. N Median 5.95 0.45 5 5.96 x 5.11 1.01

5 5.50 6.92 0.75 5 6.97
NEUTA [giga/L] day 7 Mean S.d. N Median 0.97 0.15 5 0.93 x 1.25 0.31 5 1.20 1.03 0.09 5 1.04
LYMPHA [giga/L] day 7 Mean S.d. N Median 4.77 0.43 5 5.02 x 3.61 0.86 5 3.35 5.65 0.67 5 5.65
MONOA [giga/L] day 7 Mean S.d. N Median 0.10 0.04 5 0.10 x 0.10 0.01 5 0.09 0.11 0.02 5 0.11
EOSA [giga/L] day 7 Mean S.d. N Median 0.07 0.02 5 0.06 x 0.10 0.08 5 0.06 0.07 0.01 5 0.07
BASOA [giga/L] day 7 Mean S.d. N Median 0.02 0.01 5 0.02 x 0.03 0.01 5 0.02 0.03 0.01 5 0.03
LUCA [giga/L] day 7 Mean S.d. N Median 0.03 0.01 5 0.03 x 0.02 0.01 5 0.02 0.03 0.01 5 0.03
NEUT [%] day 7 Mean S.d. N Median 16.3 2.6

5 17.2 x 24.6 4.8

5 24.9 ** 14.9 1.1

5 15.0
LYMPH [%] day 7 Mean S.d. N Median 80.1 3.2

5 79.7 x 70.5 6.1

5 70.3 * 81.6 1.3

5 81.1
MONO [%] day 7 Mean S.d. N Median 1.7 0.6

5 1.6 x 1.9 0.4

5 1.9 1.6 0.3

5 1.6
EOS [%] day 7 Mean S.d. N Median 1.1 0.4

5 0.9 x 1.9 1.3

5 1.4 1.0 0.0

5 1.0
BASO [%] day 7 Mean S.d. N Median 0.4 0.2

5 0.3 x 0.5 0.2

5 0.5 0.5 0.2

5 0.4
LUC [%] day 7 Mean S.d. N Median 0.5 0.1

5 0.5 x 0.5 0.2

5 0.6 0.4 0.1

5 0.4



### Page 89 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 4
Study 19-Dec-2011 13:58
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure White blood cell parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
WBC [giga/L] day 28 Mean S.d. N Median 6.99 1.26

5 6.27 x 7.44 0.94 5 7.30 7.06 1.37

5 6.97
NEUTA [giga/L] day 28 Mean S.d. N Median 1.39 0.92 5 1.00 x 1.12 0.26 5 1.12 1.15 0.23 5 1.18
LYMPHA [giga/L] day 28 Mean S.d. N Median 5.22 0.79 5 5.01 x 6.06 0.73 5 6.24 5.66 1.27

5 5.22
MONOA [giga/L] day 28 Mean S.d. N Median 0.12 0.03 5 0.12 x 0.11 0.06 5 0.08 0.10 0.03 5 0.10
EOSA [giga/L] day 28 Mean S.d. N Median 0.21 0.27 5 0.08 x 0.09 0.02 5 0.09 0.09 0.03 5 0.10
BASOA [giga/L] day 28 Mean S.d. N Median 0.02 0.01 5 0.02 x 0.02 0.01 5 0.02 0.02 0.01 5 0.02
LUCA [giga/L] day 28 Mean S.d. N Median 0.03 0.01 5 0.03 x 0.04 0.01 5 0.04 0.04 0.02 5 0.03
NEUT [%] day 28 Mean S.d. N Median 18.8 9.5

5 15.9 x 14.9 2.5

5 16.0 16.5 3.2

5 16.6
LYMPH [%] day 28 Mean S.d. N Median 76.0 12.6

5 80.6 x 81.5 2.8

5 80.0 79.9 3.6

5 80.2
MONO [%] day 28 Mean S.d. N Median 1.8 0.5

5 1.8 x 1.5 0.6

5 1.2 1.4 0.3

5 1.6
EOS [%] day 28 Mean S.d. N Median 2.7 3.1

5 1.4 x 1.3 0.4

5 1.1 1.4 0.5

5 1.4
BASO [%] day 28 Mean S.d. N Median 0.3 0.1

5 0.3 x 0.3 0.2

5 0.3 0.3 0.1

5 0.3
LUC [%] day 28 Mean S.d. N Median 0.4 0.1

5 0.4 x 0.5 0.1

5 0.4 0.5 0.1

5 0.5



### Page 90 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 5
Study 19-Dec-2011 14:00
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Enzymes

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
ALT [μkat/L] day 7 Mean S.d. N Median 0.80 0.14 5 0.81 x 0.80 0.07 5 0.76 0.73 0.09 5 0.68
AST [μkat/L] day 7 Mean S.d. N Median 1.79 0.25 5 1.69 x 1.98 0.19 5 1.98 2.06 0.09 5 2.06
ALP [μkat/L] day 7 Mean S.d. N Median 2.32 0.17 5 2.27 x 2.54 0.50 5 2.77 3.28 0.34 5 3.37 **

GGT_C [nkat/L] day 7 Mean S.d. N Median 8 8 5 10 x 12 4 5 10 10 5 5 10



### Page 91 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 6
Study 19-Dec-2011 14:01
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Enzymes

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
ALT [μkat/L] day 28 Mean S.d. N Median 0.82 0.10 5 0.87 x 0.89 0.11 5 0.94 0.92 0.27 5 0.77
AST [μkat/L] day 28 Mean S.d. N Median 1.55 0.17 5 1.50 x 1.61 0.20 5 1.58 1.70 0.26 5 1.71
ALP [μkat/L] day 28 Mean S.d. N Median 2.15 0.42 5 2.31 x 2.10 0.58 5 1.88 2.57 0.56 5 2.34 GGT_C [nkat/L] day 28 Mean S.d. N Median 1 2 5 0 x 0 0 5 0 3 4 5 0



### Page 92 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 7
Study 19-Dec-2011 14:02
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure
Substrates

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
UREA [mmol/L] day 7 Mean S.d. N Median 5.76 0.45 5 5.77 x 6.45 0.72 5 6.50 6.35 0.69 5 6.59
CREA [mmol/L] day 7 Mean S.d. N Median 47.0 1.7

5 46.1 x 46.1 3.4

5 46.6 45.8 8.2

5 49.2
GLUC [mmol/L] day 7 Mean S.d. N Median 5.03 0.63 5 5.35 x 5.61 0.95 5 5.46 5.56 0.72 5 5.32
TBIL [μmol/L] day 7 Mean S.d. N Median 2.50 0.52 5 2.50 x 2.00 0.33 5 1.89 2.52 0.44 5 2.34
TPROT [g/L] day 7 Mean S.d. N Median 60.28 2.49 5 59.60 x 60.79 2.96 5 60.54 61.82 1.54

5 61.01
ALB [g/L] day 7 Mean S.d. N Median 38.79 0.68 5 38.59 x 39.33 1.80

5 39.45 39.55 0.83 5 39.20
GLOB [g/L] day 7 Mean S.d. N Median 21.48 1.84

5 21.01 x 21.46 1.24

5 21.41 22.26 0.76 5 22.21
CHOL [mmol/L] day 7 Mean S.d. N Median 1.81 0.23 5 1.78 x 1.61 0.28 5 1.58 1.67 0.39 5 1.45
TRIG [mmol/L] day 7 Mean S.d. N Median 0.71 0.25 5 0.67 x 0.60 0.15 5 0.59 0.82 0.14 5 0.85



### Page 93 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 8
Study 19-Dec-2011 14:03
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure
Substrates

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
UREA [mmol/L] day 28 Mean S.d. N Median 7.18 1.09

5 6.75 x 7.49 0.95 5 7.59 8.61 0.94 5 8.42
CREA [mmol/L] day 28 Mean S.d. N Median 51.1 1.7

5 51.3 x 51.7 1.8

5 50.9 52.4 1.0

5 52.5
GLUC [mmol/L] day 28 Mean S.d. N Median 7.82 0.78 5 8.02 x 8.03 0.93 5 8.32 8.17 1.16

5 7.79
TBIL [μmol/L] day 28 Mean S.d. N Median 2.59 0.45 5 2.68 x 2.71 0.50 5 2.79 2.93 0.18 5 2.90
TPROT [g/L] day 28 Mean S.d. N Median 64.99 2.61 5 64.36 x 64.85 1.65

5 64.15 62.82 3.57 5 62.41
ALB [g/L] day 28 Mean S.d. N Median 39.37 0.54 5 39.39 x 39.70 0.56 5 39.96 38.78 1.12

5 38.47
GLOB [g/L] day 28 Mean S.d. N Median 25.62 2.63 5 24.92 x 25.14 1.23

5 24.84 24.04 2.68 5 23.96
CHOL [mmol/L] day 28 Mean S.d. N Median 2.14 0.44 5 2.00 x 1.79 0.45 5 1.60 2.02 0.44 5 2.05
TRIG [mmol/L] day 28 Mean S.d. N Median 0.78 0.33 5 0.65 x 1.05 0.56 5 1.00 0.81 0.22 5 0.74



### Page 94 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 9
Study 19-Dec-2011 14:04
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Electrolytes + minerals

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
NA [mmol/L] day 7 Mean S.d. N Median 143.2 1.3

5 142.8 x 143.2 1.1

5 143.7 142.5 2.0

5 142.9
K [mmol/L] day 7 Mean S.d. N Median 4.87 0.35 5 4.98 x 4.63 0.09 5 4.67 4.73 0.31 5 4.82
CL [mmol/L] day 7 Mean S.d. N Median 101.4 0.8

5 101.3 x 100.6 0.9

5 100.5 100.3 1.1

5 100.4
INP [mmol/L] day 7 Mean S.d. N Median 2.34 0.16 5 2.36 x 2.37 0.21 5 2.30 2.35 0.16 5 2.40
CA [mmol/L] day 7 Mean S.d. N Median 2.57 0.07 5 2.55 x 2.55 0.07 5 2.53 2.56 0.04 5 2.55
MG [mmol/L] day 7 Mean S.d. N Median 0.96 0.01 5 0.96 x 0.96 0.05 5 0.98 0.93 0.04 5 0.93



### Page 95 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 10
Study 19-Dec-2011 14:04
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Electrolytes + minerals

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
NA [mmol/L] day 28 Mean S.d. N Median 143.6 1.1

5 144.2 x 143.0 1.1

5 143.4 142.7 0.9

5 143.1
K [mmol/L] day 28 Mean S.d. N Median 4.78 0.41 5 4.59 x 4.78 0.09 5 4.78 4.85 0.19 5 4.92
CL [mmol/L] day 28 Mean S.d. N Median 103.4 1.1

5 103.3 x 101.8 1.0

5 102.3 102.4 1.8

5 102.5
INP [mmol/L] day 28 Mean S.d. N Median 2.03 0.17 5 2.02 x 1.98 0.28 5 2.05 2.05 0.27 5 2.06
CA [mmol/L] day 28 Mean S.d. N Median 2.60 0.09 5 2.62 x 2.65 0.05 5 2.63 2.61 0.12 5 2.54
MG [mmol/L] day 28 Mean S.d. N Median 0.95 0.05 5 0.92 x 0.93 0.04 5 0.91 0.90 0.03 5 0.91



### Page 96 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 11
Study 19-Dec-2011 14:06
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Acute phase proteins

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
HAPT [ng/mL] day 7 Mean S.d. N Median 338.40 221.98 5 346.00 x 463.00 117.15 5 480.00 389.40 179.32 5 427.00
A2M [ng/mL] day 7 Mean S.d. N Median 21.93 7.20 5 19.88 x 16.12 2.97 5 15.41 15.58 2.24 5 16.37



### Page 97 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 12
Study 19-Dec-2011 14:07
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Acute phase proteins

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
HAPT [ng/mL] day 28 Mean S.d. N Median 448.60 90.31 5 451.00 x 485.40 138.44 5 410.00 603.60 106.02 5 596.00
A2M [ng/mL] day 28 Mean S.d. N Median 8.92 1.96

5 7.71 x 9.45 1.33

5 9.16 8.58 1.88

5 8.65



### Page 98 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 13
Study 21-Dec-2011 07:21
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure BAL: cytology

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
TCN BAL [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 91.17 16.06 5 92.86 x+ 418.30 213.09 5 309.41 358.79 ** 355.44 156.17 5 323.33 289.85 **

MPH BAL_C [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 89.80 16.12 5 89.84 x+ 152.69 60.51 5 138.87 70.03 * 156.08 94.26 5 150.66 73.81 LY BAL_C [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 0.10 0.14 5 0.00 x+ 5.67 5.75 5 2.29 5,381.76 ** 2.97 0.90 5 3.22 2,774.36 **

PMN BAL_C [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 1.13 1.01

5 0.84 x+ 248.44 173.32 5 189.51 21,865.68 ** 177.46 67.73 5 218.75 15,590.59 **

MO BAL_C [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 0.00 0.00 5 0.00 x+ 3.81 2.69 5 3.87 ** 7.90 4.59 5 10.22 **

EO BAL_C [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 0.14 0.13 5 0.22 x+ 1.87 1.20

5 2.32 1,243.22 * 3.18 3.38 5 1.61 2,189.30 **

ATY BAL_C [cn/μL BAL] day 7 Mean S.d. N Median Deviation Vs Control 0.00 0.00 5 0.00 x+ 5.83 5.21 5 3.87 * 7.85 8.82 5 3.23 *



### Page 99 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 14
Study 21-Dec-2011 07:22
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure BAL: cytology

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
TCN BAL [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 58.56 20.39 5 60.16 x+ 75.00 21.98 5 68.55 28.07 97.65 25.65 5 107.20 66.75 MPH BAL_C [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 55.60 21.14 5 55.05 x+ 72.94 20.59 5 68.55 31.19 92.44 22.94 5 103.45 66.26 LY BAL_C [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 0.09 0.08 5 0.12 x+ 0.12 0.12 5 0.12 37.32 0.34 0.49 5 0.00 290.54 PMN BAL_C [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 2.78 1.59

5 2.98 x+ 1.91 1.84

5 2.03 -31.31 4.75 5.97 5 3.35 71.16 MO BAL_C [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 0.02 0.06 5 0.00 x+ 0.00 0.00 5 0.00 -100.00 0.06 0.14 5 0.00 156.13 EO BAL_C [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 0.06 0.10 5 0.00 x+ 0.03 0.08 5 0.00 -43.57 0.00 0.00 5 0.00 -100.00 ATY BAL_C [cn/μL BAL] day 28 Mean S.d. N Median Deviation Vs Control 0.02 0.04 5 0.00 x+ 0.00 0.00 5 0.00 -100.00 0.06 0.14 5 0.00 290.92



### Page 100 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (one-sided-), * p<=0.05, ** p <=0.01, X = Group e xcluded from statistics x=WILCOX IB 15
Study 21-Dec-2011 07:23
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure BAL: cytology

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
MPH BAL% [%] day 7 Mean S.d. N Median Deviation Vs Control 98.45 1.10

5 99.00 x- 40.00 12.04 5 45.75 -59.37 ** 43.05 11.35 5 46.50 -56.27 **

LY BAL% [%] day 7 Mean S.d. N Median Deviation Vs Control 0.10 0.14 5 0.00 x+ 1.10 0.76 5 1.00 1,000.00 * 0.90 0.22 5 1.00 800.00 **

PMN BAL% [%] day 7 Mean S.d. N Median Deviation Vs Control 1.30 1.16

5 0.75 x+ 56.25 11.47 5 49.00 4,226.92 ** 51.45 11.32 5 51.50 3,857.69 **

MO BAL% [%] day 7 Mean S.d. N Median Deviation Vs Control 0.00 0.00 5 0.00 x+ 0.85 0.38 5 1.00 ** 2.15 1.33

5 1.75 **

EO BAL% [%] day 7 Mean S.d. N Median Deviation Vs Control 0.15 0.14 5 0.25 x+ 0.60 0.45 5 0.50 300.00 * 0.75 0.50 5 0.50 400.00 *

ATY BAL% [%] day 7 Mean S.d. N Median Deviation Vs Control 0.00 0.00 5 0.00 x+ 1.20 0.72 5 1.25 * 1.70 1.51

5 1.00 *



### Page 101 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (one-sided-), * p<=0.05, ** p <=0.01, X = Group e xcluded from statistics x=WILCOX IB 16
Study 21-Dec-2011 07:24
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure BAL: cytology

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
MPH BAL% [%] day 28 Mean S.d. N Median Deviation Vs Control 94.05 3.96 5 92.50 x- 97.55 2.10 5 97.00

3.72 94.95 5.19 5 96.50

0.96
LY BAL% [%] day 28 Mean S.d. N Median Deviation Vs Control 0.20 0.21 5 0.25 x+ 0.15 0.14 5 0.25 -25.00 0.45 0.76 5 0.00 125.00
PMN BAL% [%] day 28 Mean S.d. N Median Deviation Vs Control 5.55 3.67 5 7.00 x+ 2.25 2.03 5 2.75 -59.46 4.50 4.65 5 3.50 -18.92
MO BAL% [%] day 28 Mean S.d. N Median Deviation Vs Control 0.05 0.11 5 0.00 x+ 0.00 0.00 5 0.00 -100.00 0.05 0.11 5 0.00 0.00
EO BAL% [%] day 28 Mean S.d. N Median Deviation Vs Control 0.10 0.14 5 0.00 x+ 0.05 0.11 5 0.00 -50.00 0.00 0.00 5 0.00 -100.00
ATY BAL% [%] day 28 Mean S.d. N Median Deviation Vs Control 0.05 0.11 5 0.00 x+ 0.00 0.00 5 0.00 -100.00 0.05 0.11 5 0.00 0.00



### Page 102 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 17
Study 21-Dec-2011 08:58
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure BAL: enzymes + protein

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
MTP BAL [mg/L] day 7 Mean S.d. N Median Deviation Vs Control 39 17 5 40 x+ 307 121

5 255 691 ** 565 64

5 556 1,357 **

GGT BAL_C [nkat/L] day 7 Mean S.d. N Median Deviation Vs Control 33 8 5 35 x+ 287 24

5 297 759 ** 840 121

5 772 2,416 **

LDH BAL [μkat/L] day 7 Mean S.d. N Median Deviation Vs Control 0.37 0.13 5 0.33 x+ 5.43 1.59

5 4.54 1,366.49 ** 8.36 0.63 5 8.34 2,159.46 **

ALP BAL [μkat/L] day 7 Mean S.d. N Median Deviation Vs Control 0.45 0.12 5 0.46 x+ 1.06 0.41 5 1.17 133.04 ** 2.08 0.97 5 1.58 358.15 **

NAG BAL [nkat/L] day 7 Mean S.d. N Median Deviation Vs Control 38 12 5 30 x+ 108 39

5 96 186 ** 359 28

5 376 854 **



### Page 103 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 18
Study 21-Dec-2011 10:09
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure BAL: enzymes + protein

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 1 / M Test Group 1 Micronized BATMA G 2 / M Test Group 2 Micronized BATMA
MTP BAL [mg/L] day 28 Mean S.d. N Median Deviation Vs Control 38 9 5 38 x+ 37 15 5 38 -4 54 14 5 53 41 GGT BAL_C [nkat/L] day 28 Mean S.d. N Median Deviation Vs Control 30 9 5 33 x+ 29 11 5 29 -3 56 11 5 60 86 *

LDH BAL [μkat/L] day 28 Mean S.d. N Median Deviation Vs Control 0.38 0.08 5 0.35 x+ 0.35 0.11 5 0.35 -6.38 0.54 0.19 5 0.53 44.68
ALP BAL [μkat/L] day 28 Mean S.d. N Median Deviation Vs Control 0.60 0.16 5 0.62 x+ 0.28 0.09 5 0.25 -53.64 0.32 0.10 5 0.32 -47.35
NAG BAL [nkat/L] day 28 Mean S.d. N Median Deviation Vs Control 34 7 5 39 x+ 26 8 5 23 -24 45 14 5 51 31



### Page 104 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 19
Study 21-Dec-2011 10:15
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Red blood cell + coagulation parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
RBC [tera/L] day 2 Mean S.d. N Median 8.17 0.54 5 8.40 x 8.45 0.80 5 8.36
HGB [mmol/L] day 2 Mean S.d. N Median 8.9 0.5

5 9.0 x 9.3 0.8

5 9.6
HCT [L/L] day 2 Mean S.d. N Median 0.437 0.022 5 0.446 x 0.460 0.040 5 0.467
MCV [fL] day 2 Mean S.d. N Median 53.5 1.6

5 53.8 x 54.5 2.5

5 55.5
MCH [fmol] day 2 Mean S.d. N Median 1.09 0.04 5 1.10 x 1.11 0.06 5 1.14
MCHC [mmol/L] day 2 Mean S.d. N Median 20.35 0.22 5 20.40 x 20.30 0.22 5 20.43
RET [%] day 2 Mean S.d. N Median 3.1 0.5

5 3.0 x 2.4 0.8

5 2.3
PLT [giga/L] day 2 Mean S.d. N Median 958 138

5 976 x 905 199

5 881
HQT [sec] day 2 Mean S.d. N Median 35.8 2.0

5 35.9 x 36.2 1.0

5 36.2



### Page 105 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 20
Study 21-Dec-2011 10:18
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure White blood cell parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
WBC [giga/L] day 2 Mean S.d. N Median 6.58 1.37

5 7.21 x 4.82 1.55

5 5.33
NEUTA [giga/L] day 2 Mean S.d. N Median 0.61 0.17 5 0.64 x 0.62 0.19 5 0.51
LYMPHA [giga/L] day 2 Mean S.d. N Median 5.79 1.20

5 6.15 x 4.03 1.69

5 4.71
MONOA [giga/L] day 2 Mean S.d. N Median 0.07 0.03 5 0.08 x 0.07 0.03 5 0.09
EOSA [giga/L] day 2 Mean S.d. N Median 0.06 0.07 5 0.03 x 0.05 0.04 5 0.04
BASOA [giga/L] day 2 Mean S.d. N Median 0.02 0.00 5 0.02 x 0.02 0.01 5 0.02
LUCA [giga/L] day 2 Mean S.d. N Median 0.04 0.02 5 0.03 x 0.03 0.01 5 0.03
NEUT [%] day 2 Mean S.d. N Median 9.3 1.7

5 8.3 x 16.0 13.7

5 10.8
LYMPH [%] day 2 Mean S.d. N Median 88.0 2.4

5 89.0 x 79.8 15.8

5 85.6
MONO [%] day 2 Mean S.d. N Median 1.0 0.3

5 1.1 x 1.8 1.3

5 1.6
EOS [%] day 2 Mean S.d. N Median 1.0 0.9

5 0.6 x 1.3 1.0

5 0.8
BASO [%] day 2 Mean S.d. N Median 0.3 0.1

5 0.3 x 0.4 0.1

5 0.4
LUC [%] day 2 Mean S.d. N Median 0.6 0.2

5 0.5 x 0.8 0.5

5 0.6



### Page 106 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 21
Study 21-Dec-2011 10:25
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Enzymes

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
ALT [μkat/L] day 2 Mean S.d. N Median 0.92 0.05 5 0.92 x 0.67 0.23 5 0.61
AST [μkat/L] day 2 Mean S.d. N Median 1.82 0.26 5 1.89 x 1.56 0.14 5 1.65
ALP [μkat/L] day 2 Mean S.d. N Median 3.01 0.21 5 3.03 x 2.96 0.66 5 2.61 GGT_C [nkat/L] day 2 Mean S.d. N Median 1 3 5 0 x 0 0 5 0



### Page 107 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 22
Study 21-Dec-2011 10:26
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure
Substrates

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
UREA [mmol/L] day 2 Mean S.d. N Median 4.54 0.15 5 4.50 x 5.67 1.11

5 5.41
CREA [mmol/L] day 2 Mean S.d. N Median 46.3 2.0

5 45.8 x 46.2 2.6

5 45.7
GLUC [mmol/L] day 2 Mean S.d. N Median 9.32 0.35 5 9.38 x 8.92 0.43 5 9.13
TBIL [μmol/L] day 2 Mean S.d. N Median 1.82 0.57 5 2.12 x 1.58 0.65 5 1.87
TPROT [g/L] day 2 Mean S.d. N Median 61.42 1.30

5 61.22 x 60.76 0.93 5 60.62
ALB [g/L] day 2 Mean S.d. N Median 39.02 0.70 5 39.18 x 38.26 1.49

5 38.36
GLOB [g/L] day 2 Mean S.d. N Median 22.40 0.91 5 22.64 x 22.50 0.91 5 22.26
CHOL [mmol/L] day 2 Mean S.d. N Median 1.98 0.18 5 2.05 x 2.10 0.33 5 2.02
TRIG [mmol/L] day 2 Mean S.d. N Median 0.47 0.14 5 0.39 x 0.86 0.45 5 0.75



### Page 108 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 23
Study 21-Dec-2011 10:27
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Electrolytes + minerals

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
NA [mmol/L] day 2 Mean S.d. N Median 142.1 0.6

5 142.2 x 143.9 1.0

5 144.1 *

K [mmol/L] day 2 Mean S.d. N Median 4.73 0.15 5 4.75 x 4.48 0.39 5 4.53
CL [mmol/L] day 2 Mean S.d. N Median 100.3 1.3

5 100.0 x 100.5 2.5

5 101.3
INP [mmol/L] day 2 Mean S.d. N Median 2.74 0.12 5 2.74 x 2.46 0.22 5 2.53 *

CA [mmol/L] day 2 Mean S.d. N Median 2.72 0.06 5 2.75 x 2.73 0.05 5 2.75
MG [mmol/L] day 2 Mean S.d. N Median 0.89 0.02 5 0.89 x 0.87 0.02 5 0.86



### Page 109 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 24
Study 21-Dec-2011 10:29
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Red blood cell + coagulation parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
RBC [tera/L] day 25 Mean S.d. N Median 8.13 0.32 6 8.16 x 8.01 0.63 6 7.93
HGB [mmol/L] day 25 Mean S.d. N Median 8.7 0.2

6 8.7 x 8.8 0.3

6 8.8
HCT [L/L] day 25 Mean S.d. N Median 0.419 0.011 6 0.418 x 0.424 0.012 6 0.423
MCV [fL] day 25 Mean S.d. N Median 51.5 1.0

6 51.4 x 53.1 3.3

6 53.5
MCH [fmol] day 25 Mean S.d. N Median 1.08 0.02 6 1.07 x 1.10 0.07 6 1.10
MCHC [mmol/L] day 25 Mean S.d. N Median 20.82 0.09 6 20.81 x 20.67 0.18 6 20.66
RET [%] day 25 Mean S.d. N Median 2.7 0.3

6 2.6 x 2.2 0.4

6 2.1 *

PLT [giga/L] day 25 Mean S.d. N Median 762 67

6 772 x 710 130

6 746
HQT [sec] day 25 Mean S.d. N Median 35.5 0.7

6 35.5 x 35.5 2.3

6 35.9



### Page 110 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 25
Study 21-Dec-2011 10:35
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure White blood cell parameters

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
WBC [giga/L] day 25 Mean S.d. N Median 6.10 1.17

6 5.95 x 4.94 0.68 6 5.13
NEUTA [giga/L] day 25 Mean S.d. N Median 0.96 0.23 6 0.97 x 0.87 0.20 6 0.84
LYMPHA [giga/L] day 25 Mean S.d. N Median 4.90 1.00

6 4.80 x 3.86 0.76 6 4.21
MONOA [giga/L] day 25 Mean S.d. N Median 0.10 0.03 6 0.08 x 0.10 0.03 6 0.10
EOSA [giga/L] day 25 Mean S.d. N Median 0.09 0.02 6 0.10 x 0.09 0.05 6 0.08
BASOA [giga/L] day 25 Mean S.d. N Median 0.01 0.01 6 0.01 x 0.01 0.01 6 0.01
LUCA [giga/L] day 25 Mean S.d. N Median 0.03 0.01 6 0.03 x 0.02 0.00 6 0.02 *

NEUT [%] day 25 Mean S.d. N Median 15.9 3.3

6 15.4 x 18.0 5.3

6 15.9
LYMPH [%] day 25 Mean S.d. N Median 80.1 3.5

6 81.1 x 77.5 6.3

6 80.1
MONO [%] day 25 Mean S.d. N Median 1.6 0.5

6 1.4 x 2.0 0.6

6 2.2
EOS [%] day 25 Mean S.d. N Median 1.5 0.3

6 1.7 x 2.0 1.4

6 1.6
BASO [%] day 25 Mean S.d. N Median 0.3 0.1

6 0.3 x 0.2 0.1

6 0.2 *

LUC [%] day 25 Mean S.d. N Median 0.5 0.2

6 0.5 x 0.3 0.1

6 0.3



### Page 111 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 26
Study 21-Dec-2011 10:36
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Enzymes

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
ALT [μkat/L] day 25 Mean S.d. N Median 0.69 0.11 6 0.69 x 0.78 0.19 6 0.77
AST [μkat/L] day 25 Mean S.d. N Median 1.87 0.24 6 1.89 x 1.97 0.45 6 1.85
ALP [μkat/L] day 25 Mean S.d. N Median 2.16 0.46 6 2.27 x 1.88 0.32 6 1.81 GGT_C [nkat/L] day 25 Mean S.d. N Median 6 4 6 8 x 1 2 6 0 *



### Page 112 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 27
Study 21-Dec-2011 10:38
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure
Substrates

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
UREA [mmol/L] day 25 Mean S.d. N Median 5.77 0.91 6 5.48 x 6.62 1.20

6 6.42
CREA [mmol/L] day 25 Mean S.d. N Median 52.2 3.5

6 51.8 x 55.4 2.5

6 55.8
GLUC [mmol/L] day 25 Mean S.d. N Median 6.58 0.68 6 6.57 x 6.34 0.80 6 6.37
TBIL [μmol/L] day 25 Mean S.d. N Median 2.25 0.31 6 2.25 x 2.48 0.43 6 2.60
TPROT [g/L] day 25 Mean S.d. N Median 60.88 1.12

6 60.54 x 59.79 1.90

6 59.61
ALB [g/L] day 25 Mean S.d. N Median 38.02 0.90 6 37.84 x 37.80 0.49 6 37.81
GLOB [g/L] day 25 Mean S.d. N Median 22.86 0.69 6 22.73 x 21.98 1.57

6 21.57
CHOL [mmol/L] day 25 Mean S.d. N Median 1.61 0.17 6 1.56 x 1.72 0.27 6 1.72
TRIG [mmol/L] day 25 Mean S.d. N Median 0.72 0.30 6 0.64 x 0.68 0.21 6 0.64



### Page 113 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsStatistic Profile = Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX IB 28
Study 21-Dec-2011 10:39
ToxData© System 3.0
Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Electrolytes + minerals

Sex: Male - Phase: In-life

G 0 / M Test Group 0 G 3 / M Test Group 3 Micronized BATMA
NA [mmol/L] day 25 Mean S.d. N Median 143.0 0.5

6 142.9 x 142.3 1.2

6 142.2
K [mmol/L] day 25 Mean S.d. N Median 4.60 0.25 6 4.54 x 4.63 0.20 6 4.63
CL [mmol/L] day 25 Mean S.d. N Median 102.4 1.1

6 102.2 x 102.0 1.8

6 101.4
INP [mmol/L] day 25 Mean S.d. N Median 2.17 0.14 6 2.16 x 2.14 0.11 6 2.16
CA [mmol/L] day 25 Mean S.d. N Median 2.52 0.04 6 2.50 x 2.49 0.04 6 2.50
MG [mmol/L] day 25 Mean S.d. N Median 0.91 0.02 6 0.92 x 0.92 0.06 6 0.94



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This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsInhalation Laboratory · Proj. No.: Table ID - 1 Study Means of Chamber Parameters ± Standard Deviation (SD)

mean SD mean SD mean SD mean SD mean SD mean SD mean SD
Testgruppe 0 6.0 0.0

- - 5.4 0.0 58.4 0.9 21.6 0.1 - -

- - Testgruppe 1 4.5 0.0 1.50 0.0 5.4 0.0 43.6 1.9 22.0 0.3 37 8 2.2 0.6

Testgruppe 2 4.5 0.0 1.5 0.0 5.4 0.0 43.1 0.8 21.8 0.2 133 12 10.0 1.9

Testgruppe 3 4.5 0.0 1.5 0.0 5.4 0.0 42.1 0.3 22.0 0.3 584 43 54.5 4.0

Particle size measurements

Date MMAD GSD Date MMAD GSD
Testgruppe 1 20 Sep 11 3.8 3.8 21 Sep 11 3.2 2.6 Testgruppe 2 20 Sep 11 3.4 2.2 20 Sep 11 3.4 2.3 Testgruppe 3 20 Sep 11 4.9 2.8 Concentration of substance

[mg/m3] Substance flow [mg/h] Supply air

[m3/h] Compressed air [m3/h] Exhaust air

[m3/h] Chamber humidity [%RH] Chamber temperature [°C]



### Page 133 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors

### STUDY TITLE

Report

Cobalt Lithium Manganese Nickel oxide

- Short

- term (5

- day) inhalation study in Wistar rats dust exposure

TEST FACILITY

TEST FACILITY PROJECT IDEN TIFICATION

PART II OF III ( TABLES SECTION, INDIVIDUAL VALUES )

This document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be use d only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, pub lication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory ag encies acting within the limits of their administrative authority.



### Page 134 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors

### CONTENTS OF PART II (TABLES SECTION; INDIVIDUAL VALUES)

TITL E PAGE

TABLE OF CONTENTS

CLINICAL EXAMINATIONS (Individual values) Table

Individual Signs By Interval – Clinical Observation Male animals IIA

- 1 - IIA

- 11

Individual Body W eight s Male animals IIA

- 12 - IIA

- 15

Individual Rectal Temperature Male animals IIA

- 16 - IIA

- 19

CLINICAL PATHOLOGY (Individual values)

Groups 0

- 2 (study days 7 + 28)

Red blood cell + coagulation parameters IIB

- 1 – IIB -2 White blood cell parameters IIB

- 3 – IIB -4 Enzymes IIB

- 5 – IIB -6 Substrates IIB

- 7 – IIB -8 Electrolytes + minerals IIB

- 9 – IIB

- 10 Acute phase proteins IIB

- 11 – IIB

- 12 BAL: cytology IIB

- 13 – IIB

- 16 BAL: enzymes + protein IIB

- 17 – IIB

- 18

Groups 0+3 (study day 2)

Red blood cell + coagulation parameters IIB

- 19 White blood cell parameters IIB

- 20 Enzymes IIB

- 21 Substrates IIB

- 22 Electrolytes + minerals IIB

- 23

Groups 0+3 (study day 25)

Red blood cell + coagulation parameters IIB

- 24 White blood cell parameters IIB

- 25 Enzymes IIB

- 26 Substrates IIB

- 27 Electrolytes + minerals IIB

- 28

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### PATHOLOGY (Individual values)

Absolute we ights IIC

- 1 - IIC

- 14 Relative weights IIC

- 15 - IIC

- 28 Single animal sheet IIC

- 29 - IIC

- 53 (Gross lesions and microscopic findings)

MEASUREMENTS OF EXPOSURE PARAMETERS

Exposure parameters (Daily Means) IID

- 1 - IID -4 Particle size analysis (Individual measurements) IID

- 5 - IID -9

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This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsIIA 16



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This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsInhalation Laboratory · Proj. No.: Table IID - 1 Daily Means of Exposure Parameters ± Standard Deviation (SD) Test Group 0

Date Study day

mean SD mean SD mean SD mean SD
19 Sep 2011 0 6.0 0.0 5.4 0.0 58.1 1.1 21.6 0.3 20 Sep 2011 1 6.0 0.0 5.4 0.0 58.6 2.1 21.4 0.5 21 Sep 2011 2 6.0 0.0 5.4 0.0 57.6 1.0 21.6 0.2 22 Sep 2011 3 6.0 0.0 5.4 0.0 59.9 0.6 21.6 0.2 23 Sep 2011 4 6.0 0.0 5.4 0.0 57.8 1.4 21.6 0.3

mean 6.0 0.0 5.4 0.0 58.4 1.2 21.6 0.3 SD 0.0 0.0 0.9 0.1 Chamber temperature [°C] Supply air

[m3/h] Exhaust air

[m3/h] Chamber humidity [%RH]



### Page 237 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsInhalation Laboratory · Proj. No.: Table IID - 2 Daily Means of Exposure Parameters ± Standard Deviation (SD) Test Group 1

Date Study day Substance flow [mg/h]

mean SD mean SD mean SD mean SD mean SD mean SD
19 Sep 2011 0 4.5 0.0 1.5 0.0 5.4 0.0 42.3 0.6 22.1 0.2 51 2.70 1.10 20 Sep 2011 1 4.5 0.0 1.5 0.0 5.4 0.0 42.8 1.0 22.1 0.5 34 2.70 2.00 21 Sep 2011 2 4.5 0.0 1.5 0.0 5.4 0.0 43.5 0.9 22.2 0.4 34 2.27 0.84 22 Sep 2011 3 4.5 0.0 1.5 0.0 5.4 0.0 46.9 1.1 22.2 0.1 34 1.42 0.47 23 Sep 2011 4 4.5 0.0 1.5 0.0 5.4 0.0 42.7 1.3 21.5 0.2 34 1.84 0.28

mean 4.5 0.0 1.5 0.0 5.4 0.0 43.6 1.0 22.0 0.3 37 2.2 0.9 SD 0.0 0.0 0.0 1.9 0.3 8 0.6

Particle Size Analysis

Date

MMAD
GSD Fraction of particles < 3 μm [%] 3.2
2.6

47.4 20 Sep 2011 21 Sep 2011
3.8
3.8
43.3 Chamber temperature [°C] Concentration of substance

[mg/m3] Supply air

[m3/h] Compressed air [m3/h] Exhaust air

[m3/h] Chamber humidity [%RH]



### Page 238 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsInhalation Laboratory · Proj. No.: Table IID - 3 Daily Means of Exposure Parameters ± Standard Deviation (SD) Test Group 2

Date Study day Substance flow [mg/h]

mean SD mean SD mean SD mean SD mean SD mean SD
19 Sep 2011 0 4.5 0.0 1.5 0.0 5.4 0.0 42.2 0.5 22.0 0.3 111 6.80 1.20 20 Sep 2011 1 4.5 0.0 1.5 0.0 5.4 0.0 42.5 1.3 21.9 0.4 138 11.30 0.00 21 Sep 2011 2 4.5 0.0 1.5 0.0 5.4 0.0 43.5 0.8 21.9 0.6 138 11.50 2.60 22 Sep 2011 3 4.5 0.0 1.5 0.0 5.4 0.0 44.2 0.1 21.7 0.4 138 9.78 1.10 23 Sep 2011 4 4.5 0.0 1.5 0.0 5.4 0.0 42.9 1.7 21.5 0.3 138 10.39 0.55

mean 4.5 0.0 1.5 0.0 5.4 0.0 43.1 0.9 21.8 0.4 133 9.95 1.1 SD 0.0 0.0 0.0 0.8 0.2 12 1.90

Particle Size Analysis

Date

MMAD
GSD Fraction of particles < 3 μm [%] 3.4
2.3
43.8 20 Sep 2011 20 Sep 2011

2.2
43.8 3.4 Chamber temperature [°C] Concentration of substance

[mg/m3] Supply air

[m3/h] Compressed air [m3/h] Exhaust air

[m3/h] Chamber humidity [%RH]



### Page 239 of 269 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsInhalation Laboratory · Proj. No.: Table IID - 4 Daily Means of Exposure Parameters ± Standard Deviation (SD) Test Group 3

Date Study day Substance flow [mg/h]

mean SD mean SD mean SD mean SD mean SD mean SD
19 Sep 2011 0 4.5 0.0 1.5 0.0 5.4 0.0 41.9 0.4 22.2 0.3 614.0 57.3 0.8 20 Sep 2011 1 4.5 0.0 1.5 0.0 5.4 0.0 42.3 0.8 21.8 0.5 553.0 51.7 7.9

mean 4.5 0.0 1.5 0.0 5.4 0.0 42.1 0.6 22.0 0.4 584 54.5 4.4 SD 0.0 0.0 0.0 0.3 0.3 43 4.0

Particle Size Analysis

Date

MMAD
GSD Fraction of particles < 3 μm [%]

Due to lethality occurred, only two exposure was performed. 2.8
31.5 4.9 20 Sep 2011 Chamber temperature [°C] Concentration of substance

[mg/m3] Supply air

[m3/h] Compressed air [m3/h] Exhaust air

[m3/h] Chamber humidity [%RH]



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### Table IID

- 5 Particle size analysis by cascade impactor, test group 1/1

Table IID

- 6 Particle size analysis by cascade impactor, test group 1/2

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### Table IID -7 Particle size analysis by cascade impactor, test group 2/1

Table IID

- 8 Particle size analysis by cascade impactor, test group 2/2

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### Table IID -9 Particle size analysis by cascade impactor, test group 3/1

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### STUDY TITLE

Report

Cobalt Lithium Manganese Nickel oxide

- Short

- term (5

- day) inhalation study in Wistar rats dust exposure

TEST FACILITY

TEST FACILITY PROJECT IDEN TIFICATION

PART III OF III ( SUPPLEMENT )

This document contains manufacturing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be use d only for that purpose for which it was intended by the Sponsor(s). Every other or additional use, exploitation, reproduction, publication or submission to other parties require the written permission of the Sponsor(s), with the exception of regulatory agencies acting within the limits of their administrative authority.



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### CONTENTS OF PART I I I ( SUPPLEMENT )

TITLE PAGE

TABLE OF CONTENTS

Analysis of the test substance

Characterization of the test substance (GKC Auftrag 12115)

Historical control data of clinical pathology testing

Analysis of urine samples

Analysis of lungs

Analysis of extra

- pulmonary organs

Analysis of blood

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