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 IDENTIFICATION

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 used 

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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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 LABORATORY 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 AUTHORITY)

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CONTENTS

TITLE PAGE

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

BLANK PAGE FOR EPA SUBMISSION ONLY

FLAGGING CRITERIA

SIGNATURE PAGE

CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL

STATEMENT OF THE QUALITY ASSURANCE UNIT

GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY)

CONTENTS

1. SUMMARY

2. INTRODUCTION

2.1. OBJECTIVES

2.2. SELECTION OF CONCENTRATIONS

2.3. TEST GUIDELINES

2.4. TIME SCHEDULE

2.5. RETENTION OF RECORDS

2.6. ANIMAL WELFARE

3. MATERIALS AND METHODS

3.1. TEST ITEM

3.2. TEST SYSTEM

3.3. HOUSING AND DIET

3.4. TEST GROUPS AND TARGET CONCENTRATIONS

3.5. GENERATION OF THE INHALATION 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 PROCEDURE

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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 EXAMINATIONS

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 EXAMINATIONS

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 ABBREVIATIONS

8.1. CLINICAL EXAMINATIONS

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 Body Weights

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 groups) 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-12 – IC-14

Incidence and grading of microscopic findings IC-15 - IC-18 

MEASUREMENTS OF EXPOSURE PARAMETERS (Summary table)

Exposure Parameters 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 Weights

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 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

Exposure 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/m3

. 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:

As 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 

Mean SD (μm)

1 2 2.2 0.6 3.2 and 3.8

2 10 10.0 1.9 3.4 and 3.4

3 50 54.5 4.0 4.9

The following substance-related adverse effects were observed:

Test group 3 (50 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 

• Degeneration/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 haematocrit 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 haematocrit 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/m3 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 systemically available, urine sample of the high concentration group animals were 

collected after the last exposure. Lung burden of the test material was determined in satellite 

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 CONCENTRATIONS

As a range finding study with a potentially poorly soluble particular matter with expected low 

pulmonary toxicity the concentrations are chosen based on available data of pigmentary 

TiO2. The following concentrations were selected for the present study:

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

10 mg/m3: as the mid concentration expected to cause 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 2011

Experimental starting date (Arrival of test 

animals):

06 Sep 2011

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: Study 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 evaluation

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 relevant 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 METHODS 

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) refers 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 2011 

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 cm2

). 

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 by 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 from these ranges did not occur.

The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. The floor and the walls 

were cleaned once a week with water containing an appropriate disinfectant.

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 TARGET 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. Due 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/m3) 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/m3) 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/m3) 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/m3) 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/m3) 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/m3) 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

47a x

48a x

49a x

50 x x

51 x x x

52b x x

53 x x x

54a x

55a x

56a x

121a x x

122c x x

123 x

124c x x

125a x x

126a x x

127a x x

128a 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

 Sacrificed moribund on study day 3, necropsied as soon as possible

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3.5. GENERATION OF THE INHALATION 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 

compressed 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 – 200 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

The 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. Determination 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 systems were continuously 

monitored using scattered light photometers.

The analyses were carried out at the 

3.6.2.1. Sampling for gravimetric 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 filter 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, with reference to the sample 

volume of the inhalation atmosphere.

3.6.2.3. Real time monitoring of constancy of concentrations

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 ratio 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, Billerica, MA 01821, USA) 

• Sampling probe internal diameter 7 mm 

• Balance Sartorius M3P-000V001 and LC 1201S-00MS (Sartorius AG, Göttingen, 

Germany)

Sampling for particle size analyses:

Preweighed metal collecting discs and a backup particle filter were placed into the cascade 

impactor: One 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:

Gravimetrical 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 size 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 PROCEDURE

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 among 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 

minimize 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 nominal 

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: t99 (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 system

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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 EXAMINATIONS 

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 individual differences.

Due to clinical signs of toxicity and lethality observed in test group 3, body 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 were 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 repeated again starting from 

study day 17 until study day 18. Aliquots of the urine were examined for creatinine 

concentration at 

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3.8.5. Rectal temperature

On study day 2, rectal temperature was determined in all surviving animals. For animals 

exposed on this day, the measurement was performed after exposure. 

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

On study day 4, rectal temperature was 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 was performed after exposure.

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3.9. LUNG BURDEN 

Due to premature death of group 3 animals, the lungs 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 stored 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 organs and tissues 

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 

carried 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-K3 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, 6th 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, wavelength 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-γglutamyl-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

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Blood 

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 dehydrogenase 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/ glycerol3-phosphate oxidase/4-aminophenazone

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 xylidylblue 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 α2-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--glutamyltransferase; 

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-NADoxidoreduktase; 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-phosphoric 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-glucosideacetamidodeoxy-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 extends 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 died 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 histopathology 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 (modified 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 solution.

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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 organs and tissues for histopathology

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. Tracheaf 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 comparison 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 For parameters with 

bidirectional changes: 

Non-parametric one-way 

analysis using KRUSKALWALLIS 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 (two-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)

Nominal 

concentration 

Mean SD (mg/m3)

0 -

1 2 2.2 0.6 6.2

2 10 10.0 1.9 22.0

3 50 54.5 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 are 

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 protocols 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 means of relative humidities in the inhalation systems ranged between 41.9 and 

59.9 %. Daily means 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, the calculated mass fractions of particles below 3 μm 

aerodynamic 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 the 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/m3) 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

3.27 / 2.10

3.09 / 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*105

/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 EXAMINATIONS 

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)

Lethality 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 in a moribund state on study day 3. 

Total 16 of the 22 animals were found dead or sacrificed in a moribund state within the first 

days.

4.2.2. Clinical observations

(Summary tables 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 sacrificed in 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 from study day 0 to 4: group 3 

Significantly increased from 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 temperatures 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 days 3 

and 4.

Figure 7: Mean rectal temperature of male animals 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 ions. 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 30 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/m3) at study day 7 and 

after a 3 weeks recovery period at study day 28. In rats of test group 3 (50 mg/m3) 

venipuncture was performed at study day 2 and again after a 3 weeks recovery period at 

study day 25.

Broncho-alveolar lavage fluid was sampled in rats of test groups 1 and 2 (2 and 10 mg/m3), 

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/m3) red blood cell (RBC) counts, 

hemoglobin and haematocrit values were higher compared to controls. 

At study day 7, in rats of test group 1 and 2 (2 and 10 mg/m3) 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/m3

) 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/m3) 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 counts 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/m3) alkaline phosphatase activities were 

increased.

At study day 2, in rats of test group 3 (50 mg/m3) 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/m3

) 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 treatmentrelated.

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/m3

Gr. 2

10 mg/m3

Gr. 1 

2 mg/m3

Gr. 2

10 mg/m3

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/m3) 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. 

Absolute 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/m3

Gr. 2

10 mg/m3

Gr. 1 

2 mg/m3

Gr. 2

10 mg/m3

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/m3) total protein, γglutamyl transferase (GGT), and lactate dehydrogenase (LDH) activities were moderately 

increased. Alkaline phosphatase (ALP) and β-N-acetyl glucosaminidase (NAG) activities 

were marginally increased in rats of test group 1 (2 mg/m3) and moderately increased in 

those of test group 2 (10 mg/m3). 

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/m3) 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 group 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 lungs 

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 epithelium 

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 lungs 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 lymphoreticulocellular7

• 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 findings 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 aerosols were 

respirable for rats. 

Inhalation exposure of 50 mg/m3 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 hypothermia. 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. On 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 exposures 

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 detection limit. Extrapulmonary organs were only examined 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, organ 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 of 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 Co2+ 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 indication 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/m3) a hemo-concentration took place, indicated by higher red 

blood cell counts, haematocrit and hemoglobin values. The reason of a higher alkaline 

phosphatase activity in rats of the higher test group cannot be elucidated, but a treatmentrelated 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, LDH 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 lesions (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 were observed in all animals. 

In the 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, 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 

of 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/m3 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. 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 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. 

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. 1st 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. 2009. Nov;61(6):591-603.

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8. LIST OF ABBREVIATIONS 

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 EXAMINATIONS 

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 = Head-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/m3 = 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 = α2-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 = 1012/liter

giga/L = giga/liter = 109 /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).

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IA- 1

Study 

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary - Clinical Observation 

Sex: Male - Phase: In-life 

G 0 / M 

Test Group 0

G 1 / M 

Test Group 1

G 2 / M 

Test Group 2

G 3 / M 

Test Group 3

day -4 -> -3

Animals examined N 22 22 22 22

normal NAD

N 22 22 22 22

% 100.0 100.0 100.0 100.0

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IA- 2

Study 

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary - Clinical Observation 

Sex: Male - Phase: In-life 

G 0 / M 

Test Group 0

G 1 / M 

Test Group 1

G 2 / M 

Test Group 2

G 3 / M 

Test Group 3

day 0 [BE] -> day 4 [AE]

Animals examined N 22 22 22 22

dead N6 6 6 16

% 27.3 27.3 27.3 72.7

found dead N0 0 0 14

% 0.0 0.0 0.0 63.6

sacrificed scheduled N6 6 6 0

% 27.3 27.3 27.3 0.0

sacrificed moribund N0 0 0 2

% 0.0 0.0 0.0 9.1

normal NAD

N 22 22 22 22

% 100.0 100.0 100.0 100.0

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IA- 3

Study 

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary - Clinical Observation 

Sex: Male - Phase: In-life 

G 0 / M 

Test Group 0

G 1 / M 

Test Group 1

G 2 / M 

Test Group 2

G 3 / M 

Test Group 3

day 7 -> 28

Animals examined N 16 16 16 6Animals with signs N0 0 0 5 % 0.0 0.0 0.0 83.3dead N 16 16 16 6 % 100.0 100.0 100.0 100.0found dead N0 0 0 0 % 0.0 0.0 0.0 0.0sacrificed scheduled N 16 16 16 6 % 100.0 100.0 100.0 100.0sacrificed moribund N0 0 0 0 % 0.0 0.0 0.0 0.0respiration N0 0 0 5 % 0.0 0.0 0.0 83.3accelerated N0 0 0 5 % 0.0 0.0 0.0 83.3intermittent N0 0 0 2 % 0.0 0.0 0.0 33.3fur piloerection

N0 0 0 1% 0.0 0.0 0.0 16.7

normal NAD

N 16 16 16 6% 100.0 100.0 100.0 100.0

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Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics 

n=DUNNETT

IA- 4 

Study 

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary Body Weights - BW / Body Weights [g] 

Sex: Male - Phase: In-life 

G 0 / M

Test Group 0

G 1 / M 

Test Group 1 

G 2 / M

Test Group 2

G 3 / M

Test Group 3

day -4 Mean 

S.d. N Deviation Vs Control 

248.0

9.5

22

n 246.1 

9.3 

22 

-0.8 

247.1

9.0

22

-0.4

247.7

12.3

22

-0.2

day 0 Mean 

S.d. N Deviation Vs Control 

257.9

9.0

22

n 258.8 

11.8 

22 

0.3 

257.8

12.5

22

-0.1

258.0

16.2

22

0.0

day 2 Mean 

S.d. N Deviation Vs Control 

255.1

10.8

22

n 257.1 

12.6 

22 

0.8 

255.8

13.9

22

0.3

230.4

14.5

22

-9.7

** 

day 3 Mean 

S.d. N Deviation Vs Control 

0

X 

0 

X 

0

X 220.8

14.4

11

X 

day 4 Mean 

S.d. N Deviation Vs Control 

256.9

11.0

22

n 260.2 

13.1 

22 

1.3 

254.4

14.8

22

-1.0

238.5

16.2

6

-7.2

* 

day 7 Mean 

S.d. N Deviation Vs Control 

262.0

14.3

16

n 261.1 

15.0 

16 

-0.3 

256.5

18.1

16

-2.1

255.5

17.4

6

-2.5

day 11 Mean 

S.d. N Deviation Vs Control 

283.9

9.7

11

n 281.7 

16.6 

11 

-0.7 

275.0

18.5

11

-3.1

273.1

18.7

6

-3.8

day 14 Mean 

S.d. N Deviation Vs Control 

297.9

9.3

11

n 294.8 

18.4 

11 

-1.0 

290.9

18.1

11

-2.4

287.0

20.3

6

-3.6

day 18 Mean 

S.d. N Deviation Vs Control 

300.1

20.4

11

n 312.9 

21.1 

11 

4.3 

294.2

23.7

11

-2.0

304.9

21.3

6

1.6

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Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics 

n=DUNNETT 

IA- 5

Study 

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary Body Weights - BW / Body Weights [g] 

Sex: Male - Phase: In-life 

G 0 / M

Test Group 0

G 1 / M 

Test Group 1 

G 2 / M

Test Group 2

G 3 / M

Test Group 3

day 21 Mean 

S.d. N Deviation Vs Control 

321.3

11.1

11

n 324.3 

23.5 

11 

0.9 

313.4

20.2

11

-2.4

314.2

22.2

6

-2.2

day 25 Mean 

S.d. N Deviation Vs Control 

319.0

16.1

11

n 325.5 

30.2 

11 

2.1 

316.5

21.5

11

-0.8

303.7

21.7

6

-4.8

day 28 Mean 

S.d. N Deviation Vs Control 

329.1

4.9

5

n 335.9 

21.5 

5 

2.0 

322.3

20.9

5

-2.1

0

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Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics 

d = day; n=DUNNETT 

IA- 6

Study 

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary Changes Body Weights - BW / Body Weights [g] 

Sex: Male - Phase: In-life 

G 0 / M

Test Group 0

G 1 / M

Test Group 1

G 2 / M

Test Group 2

G 3 / M

Test Group 3

d -4 -> 0 Mean 

S.d. N Deviation Vs Control 

9.93.4

22

n 12.7

4.2

22

27.9

10.7

6.5

22

7.8

10.3

4.9

22

4.5

d 0 -> 4 Mean 

S.d. N Deviation Vs Control 

-1.1

5.1

22

n 1.4

4.7

22

-226.8

-3.4

5.1

22

213.4

-12.3

8.2

6

1,029.1

** 

d 7 -> 11 Mean 

S.d. N Deviation Vs Control 

14.7

3.3

11

n 15.5

4.6

11

5.7

13.7

5.2

11

-6.9

17.6

3.6

6

19.5

d 14 -> 18 Mean 

S.d. N Deviation Vs Control 

2.3

15.0

11

n 18.1

3.5

11

698.4

** 3.3

13.4

11

45.6

17.9

3.3

6

687.6

* 

d 21 -> 25 Mean 

S.d. N Deviation Vs Control 

-2.3

17.5

11

n 1.3

13.7

11

-155.8

3.1

12.7

11

-235.1

-10.5

2.5

6

359.4

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Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics 

n=DUNNETT 

IA - 7

Study 30-Apr-2013 11:51

ToxData© System 3.0

Short-term (5-day) inhalation toxicity study in male Wistar rats dust exposure Summary Rectal Temperature (°C) 

Sex: Male - Phase: In-life

G 0 / M

Test Group 0

G 1 / M

Test Group 1

G 2 / M

Test Group 2

G 3 / M

Test Group 3

day 2 Mean 

S.d. N 

37.6

0.5

22

n 37.3

0.7

22

37.4

0.5

22

35.5

1.7

13

** 

day 3 Mean 

S.d. N 0

X 

0

X 

0

X 37.3

1.8

8

X 

day 4 Mean 

S.d. N 

37.3

0.4

5

n 37.9

0.5

5

36.9

0.3

5

36.7

0.4

6

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Statistic 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

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Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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

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Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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

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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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (one-sided-), * p<=0.05, ** p <=0.01, X = Group excluded 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 sponsors
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (one-sided-), * p<=0.05, ** p <=0.01, X = Group excluded 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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 sponsors
Statistic 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

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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

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Statistic 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

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Statistic 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

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Statistic 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

* 

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Statistic 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

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Statistic 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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Inhalation 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]

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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 IDENTIFICATION

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 used 

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 II (TABLES SECTION; INDIVIDUAL VALUES)

TITLE PAGE

TABLE OF CONTENTS

CLINICAL EXAMINATIONS (Individual values) Table

Individual Signs By Interval – Clinical Observation

Male animals IIA-1 - IIA-11

Individual Body Weights

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 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

Exposure parameters (Daily Means) IID-1 - IID-4 

Particle size analysis (Individual measurements) IID-5 - IID-9 

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IIA 16

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Inhalation 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]

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Inhalation 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]

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Inhalation 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]

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Inhalation 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 IDENTIFICATION

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 used 

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 III (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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