Single Wall Carbon Nanotubes (SWCNT)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Aug 2014 - 31 Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

adopted in 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

adopted in 2009

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

adopted in 1998

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

943-098-9

Cas Number:

7440-44-0

Molecular formula:

Carbon. Morphological features: - Tube cylindrical surface is formed by 6-membered rings consisting of carbon atoms linked by a double bond. - Carbon nanotube chirality is random. - Tube length: 1 - 10 μm. - Agglomeration: CNTs tend for form bundles

IUPAC Name:

carbon

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHanTM: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Horst, Netherlands
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 213.2 - 259.8 g (mean 236.6 g), 143.4 - 179.7 g (mean 163.2 g)
- Housing: In groups of four in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment.
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 20/14 and 46/14) rat / mouse maintenance diet (Provimi Kliba AG, Kaiseraugst / Switzerland), ad libitum (except for the period when the animals were restrained in the exposure tubes and before blood sampling for clinical laboratory investigation)
- Water: Tap water, ad libitum (except for the periods when the animals were restrained in the exposure tubes)
- Acclimation period: 6 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Aug 2014 To: 10 Feb 2015

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 11.19 - <= 14.59 µm

Geometric standard deviation (GSD):

8.78

Remarks on MMAD:

The MMAD was above the target range. Nevertheless the MMADs were considered to be appropriate for this type of study and characteristics of the test item, especially as an MMAD for carbon nano tubes is not an appropriate measurement.

GSD was 8.78 and 5.48 for the 0.5 and 5 mg/m³ air group, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes.
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating particulates/aerosols: not reported
- Temperature, humidity, pressure in air chamber: The mean emperature was between 21.9 and 22.8 °C, the mean relative humidity level ranged from 3.3 - 3.7%, and the mean oxygen concentration was 20.8%.
- Air flow rate: 1 L/min
- Air change rate: not reported
- Method of particle size determination: The cumulative particle size distribution of the test aerosol was determined using a cascade impactor. The test aerosol was impacted at each stage onto an appropriate medium (covered with grease) and the particle size distribution of the test item in the generated aerosol was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor five times for groups 3 and eight times for group 4. The impactor for group 3 was taken over a period of 6 days. Due to the low aerosol concentration, impactor sampling was not possible for group 2.
- Treatment of exhaust air: The exhaled air is exhausted through the gap near each feed tube out of the exposure chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: The test item usage (nominal aerosol concentration) was measured once per day in group 4 by weighing the piston containing the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration.
Gravimetric determination of the aerosol concentration was performed once per exposure for groups 2 and 4 and twice per exposure for group 3. The duration of sampling was sufficient to ensure reliable results. One filter per exposure was collected during the majority of the 6-hour exposure period. Additional monitoring filters were taken for group 4 at shorter intervals for adjusting the aerosol concentration during exposure (if necessary) and retained in the raw data.
Test aerosol samples were collected onto a suitable filter using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port except for the filter taken at the pipe for group 2. For this filter, an air-flow rate of approximately 3.75 L/min was used. The filters were weighed before and immediately after sampling using a calibrated balance.
The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test article concentration and particle size distribution were analyzed in air. The method to analyze both is described under "Details on inhalation exposure". It was found that the actual achieved test article concentration was 99.7 - 105.3% of the target concentration. The MMAD was with up to 14.59 µm above the range of 1 to 3 μm for the measured groups. Therefore deposition of the particles was considered to have occurred mainly in the upper respiratory tract. Only a small portion of the particles was considered to be able to reach the lower respiratory tract. Nevertheless the MMADs were considered to be appropriate for this type of study and characteristics of the test item.

Duration of treatment / exposure:

6 h daily for 13 weeks

Frequency of treatment:

5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.05 mg/m³ air (nominal)

Remarks:

the gravimetrically determined aerosol concentration was 0.051 mg/m³ air.

Doses / concentrations 2

Dose / conc.:

0.5 mg/m³ air (nominal)

Remarks:

the gravimetrically determined aerosol concentration was 0.527 mg/m³ air.

Doses / concentrations 3

Dose / conc.:

5 mg/m³ air (nominal)

Remarks:

the gravimetrically determined aerosol concentration was 4.99 mg/m³ air.

No. of animals per sex per dose:

15 (10 main study, 5 BAL analysis)
plus 15 for recovery groups in the vehicle and high dose group. (10 for recovery, 5 for BALF analysis after recovery).

Please refer to Table 1 under "Any other information on materials and methods incl. tables" for details.

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes (18 h)
- Rationale for selecting satellite groups: recovery was analyzed in the high dose and vehicle control group.
- Post-exposure recovery period in satellite groups: 13 weeks

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily before and after exposure, once daily on weekends, acclimatization and recovery.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly (each individual animal) during the first 4 weeks of treatment, weekly thereafter and weekly during acclimatization and recovery.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimatization, in week 13 of treatment and at the end of the recovery period
- Dose groups that were examined: All main study animals of all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In week 13 of treatment and at the end of the recovery period (both times early in the working day)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes (18 h in metabolism cages but with access to water)
- How many animals: All animals of all groups
- Parameters checked: Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index (low, medium, high fluorescence), leukocyte count, total, differential leukocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells, platelet count), prothrombin time (= thromboplastin time), activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In week 13 of treatment and at the end of the recovery period (both times early in the working day)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes (18 h in metabolism cages but with access to water)
- How many animals: All animals of all groups
- Parameters checked: Glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transferase, creatine kinase, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, globulin, albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: 18 h
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food only, 18 h)
- Parameters checked: Urine volume, specific gravity (relative density), color, appearance, pH value, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All animals of all groups
- Battery of functions tested: Functional Observation Battery (FOB): Animals of allocation A (see table under "Any other Information on materials and methods incl. tables") were observed for behavior, reflexes, activity, responsiveness, urine or feces, posture and general abnormalities once at the end of the treatment period. Grip strength was measured. Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with a suitable device. Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes are reported.

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Animals were sacrificed approximately 22 - 26 h after the last exposure or at the end of the recovery period.
- Dose groups that were examined: All groups including recovery and control
- Number of animals: 5/sex/group
- Parameters checked: Enzymatic activity of lactate dehydrogenase, alkaline phosphatase, and total protein (storage at 2 - 8 °C until analysis), from the lavage fluid, cell suspensions were prepared and the cells were counted (total and differntial cell count including macrophages, neutrophils, lymphocytes, eosinophils, epithelial cells, and other cells), two pieces of smears per animal were prepared, stained with Diff-Quick and 500 cells per smear were counted by light microscopy.

LUNG BURDEN: Yes
- Time schedule for analysis: After necropsy
- Dose groups that were examined: All dose groups
- Parameters checked: The tracheobronchial lymph node and the right medial lung lobe were weighed, underwent low temperature ashing and the ashes underwent EDX analysis and/or element mapping to detect the test item and/or RAMAN analysis and/or powder diffraction X-ray analysis to detect the test item. The BALF smears were evaluated by laser scanning microscopy to detect the test item. The smear may undergo EDX analysis and/or element mapping. The remaining formalin fixed lung left lobe tissue were sliced manually into slices of approximately 1 - 2 mm in thickness (maximum two slices) and the slices were dried for 20 days at 25 ºC and thereafter the presence of test item was evaluated by EDX analysis and/or element mapping to detect the test item and/or RAMAN analysis.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All allocation A and C animals (see table under "Any other Information on materials and methods incl. tables") surviving to the end of the observation periods were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. The animals that died spontaneously were transferred to the pathology unit on the same day and necropsied immediately.

The following organs were weighed before fixation: Adrenal glands, brain, epididymides, heart including auricles, kidneys, liver, lungs, ovaries, lymph nodes tracheobronchial, spleen, testes, thymus, uterus with vagina, uterine cervix and oviducts

HISTOPATHOLOGY: Yes (see table 2 for details)
- Fixative: Neutral phosphate buffered 4% formaldehyde solution
- Embedding media: Paraffin
- staining: Haematoxylin-eosin (H&E)
- Tissues analyzed from groups: For the allocation A and C animals (see table under "Any other Information on materials and methods incl. tables") of the groups 1 (vehicle) and 4 (high dose) and any animal found dead before the planned day of sacrifice. Sections of the lungs from all allocation A and C animals of all groups were prepared and examined. Additional tissue (muzzle) was processed and examined in an animal found dead during the treatment period.
Any gross lesion that was macroscopically identified was examined microscopically in all animals.

Statistics:

The following statistical methods were used to analyze food consumption, body weight, clinical
laboratory data, ophthalmoscopic examinations, grip strength, locomotor activity, BALF, organ
weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance
estimate was applied if the variables could be assumed to follow a normal distribution
for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs considered to be related to treatment with the test item.
Decreased activity and ruffled fur were recorded in the control animal that died prematurely during the treatment period.
Findings, such as scabs, nodule, hair loss and injury, were noted in single animals and therefore considered to be incidental. An open mass was recorded during the recovery period in the control animal that died prematurely.

Summarized results can be found in Attachment 1.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female of the vehicle group was found dead on Day 59 of treatment and an additional female of the vehicle group was found dead on Day 66 of recovery.
All other animals survived the scheduled treatment or recovery periods.

Summarized results can be found in Attachment 1.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on body weights and body weight gains in any group during the treatment and recovery periods.
Some values occasionally achieved statistical significance but did not show a relationship to dosage.

Summarized results can be found in Attachment 1.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on food consumption during the treatment and recovery periods.
Slight, occasionally statistically significant, reductions in food intake were recorded during the treatment period in females of the high dose groups. As this was only slight and occurred transiently, it was considered to be incidental.

Summarized results can be found in Attachment 1.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no ophthalmoscopic findings considered to be related to treatment with the test item at the end of the treatment or recovery periods.
The findings noted were considered to be incidental as they are commonly seen in animals of this age and strain and are recorded in all groups including controls.

Summarized results can be found in Attachment 1.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in haematology parameters considered to be related to treatment with the test item.
Some differences achieved statistical significance at the end of the treatment period (e.g. relative reticulocyte count in males of the 0.5 mg/m³ group, relative reticulocyte count and basophils in females of the 0.5 mg/m³ group, white blood cell count and lymphocytes in females of the 0.5 mg/m³ and 5 mg/m³ group, haemoglobin concentration distribution width in females of the 5 mg/m³ group, monocytes and platelets in females of the 5 mg/m³ group) or at the end of the recovery period (e.g. basophils, large unstained cells and monocytes in males of the 5 mg/m³ group, haemoglobin and haematocrit in females of the 5 mg/m³ group). These changes were either in the intermediate groups or very slight and within the historical control range and, therefore, considered not toxicologically relevant.

Summarized results can be found in Attachment 1 and Table 3 under "Any other information on results incl. tables".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in clinical biochemistry considered to be related to treatment with the test item.
Some differences achieved statistical significance at the end of the treatment period (e.g. creatinine, phosphorus and potassium in males of the low dose group, A/G ratio in females of the low dose group, potassium in females of the mid dose group) or at the end of the recovery period (e.g. protein and albumin in males of the high dose group), but these were either in the intermediate groups or still within the historical control range and, therefore, considered not toxicologically relevant.

Summarized results can be found in Attachment 1 and Table 4 under "Any other information on results incl. tables".

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: The weight of liver, kidneys, ovaries, testis, adrenal glands, brain and uterus and the histopathology of uterus, vagina, cervix, epididymis, ovaries, prostate, seminal vesicles incl. coagulation glands, testis, thyroid, adrenals, brain and pituitary. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in urinalysis parameters considered to be related to treatment with the test item.
A slight decrease in pH was recorded in males of the high dose group at the end of the recovery period but this was considered not toxicologically relevant.

Summarized results can be found in Attachment 1.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in organ weights after 13 weeks of treatment and after 13 weeks recovery considered to be related to treatment with the test item.
The statistically significant spleen weight in females of the low dose group at the end of treatment and kidneys and uterus in females of the high dose group at the end of recovery did not show a relationship to dose or were considered biological variation without any microscopic correlation.

Summarized results can be found in Attachment 1 and Table 5 under "Any other information on results incl. tables".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings in males after 13 weeks of treatment or 13 weeks recovery considered to be related to treatment with the test item.
Enlarged and discolored bronchial lymph nodes recorded in one female of the high dose group at the end of treatment was considered incidental. This was based on the microscopic correlation of activation and blood absorption/erythrophagocytosis which are not uncommon findings in rats of this strain and age. All other findings noted are commonly seen in animals of this age and strain and are therefore, considered to be incidental.

Summarized results can be found in Attachment 1.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no effects on mean functional observational battery considered to be related to treatment with the test item.
During the functional observational battery in week 13, reduction in the number of rearings was recorded in males treated with the test item. As it was also seen in males of the control group and there was no relationship to dosage in females, it was considered to be unrelated to treatment with the test item.

There were no effects on grip strength considered to be related to treatment with the test item.
The mean fore- and hind limb grip strength values of the test item-treated animals compared favorably with those of the respective control animals.

There were no effects on mean locomotor activity considered to be related to treatment with the test item.
Reductions seen in mean locomotor activity of the females of the 0.5 mg/m³ air group and both sexes of the high dose group were considered to be of uncertain toxicological relevance.

Summarized results can be found in Attachment 1.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Most microscopic findings recorded were of agonal, incidental or spontaneous characters, and those were within the range of normal background lesions which may be recorded in toxicology studies using animals of this strain and age, and hence, were considered to be not related to treatment with the test item.

The only treatment-related finding were macrophages of the lung phagocytizing blackish material (test item). This was observed in all test groups in a dose-dependent manner. In addition, intra-alveolar conglomeration of macrophages phagocytizing blackish material were observed at a minimum severity in both sexes of the mid and high dose group, and the deposit of blackish material in the interstitium at bronchiolar-alveolar area was recorded at a minimum severity in both sexes of the high dose group. However, as far as the lung tissue was examined qualitatively, the amounts of macrophages in the lung of animals from the test item treatment groups were within the range of that from the controls, and no obvious increase in the number of macrophages were identified in any animals of any treatment group. In any animals examined, except for the above-mentioned findings, there was neither further evidence of inflammatory responses nor other degenerative and/or proliferative lesions that could be attributable to treatment with the test item.
In the recovery animals, macrophages phagocytizing blackish material were also observed in all BALF smear samples of the high dose group (allocation D), but its ratio (%) to total number of cells counted decreased compared to the ratio obtained in the non-recovery animals. In the histologic examination, the group mean severity of macrophages phagocytizing blackish material decreased, but macrophages phagocytizing blackish material were still present in all animals of the high dose group.
The incidence and group mean severity of the remainder of findings were not different between the non-recovery and the recovery of the high dose group.
The above-mentioned findings obtained under the condition of this study were within the range of normal physiological responses for scavenging foreign materials. No further relevant findings were observed in any animals from non-recovery and recovery groups, and therefore, all findings were considered not to be adverse.

Summarized results can be found in Attachment 3 and Table 6 under "Any other information on results incl. tables".

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Analysis of BALF
After 13 weeks of treatment there were no changes in viability and cell count and no effects on the enzymatic activity of protein and alkaline phosphatase levels. Lactate dehydrogenase level was slightly, but statistically significantly increased in females of the high dose group at the end of the treatment period. This increase was not present at the end of the recovery period.
In the differential cell count, macrophages phagocytizing blackish material (test item) were observed in all smear samples of the test item treated groups in a dose dependent manner at the end of the treatment period. Most macrophages phagocytizing blackish material existed alone, but macrophages showing multinucleated syncytial giant cell appearance were observed also on
smears from the middle and high dose group. There were no toxicologically significant differences in the ratio in the remainder of cell types including eosinophils, neutrophils, lymphocytes, epithelial cells and others.
At the end of the recovery period, macrophages phagocytizing blackish material were also observed in all BALF smear samples of the high dose group, but the ratio to total number of cells counted decreased compared to the ratio obtained in the non-recovery animals.
Pigment laden macrophages seen in two females of the control group was considered to be an ex vivo contamination as the finding appeared in only two animals and all control animals were exposed together in a separate exposure system.

Summarized results can be found in Attachment 1 and Table 7 and 8 under "Any other information on results incl. tables".

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

5 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to the highest concentration tested.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 3:    Haematology findings following 90 days of treatment in rats

Dose level	Males				Females
mg/m³	0	0.1	0.5	5	0	0.1	0.5	5
Relative reticulocyte count
Treatment phase Day 90 (% to control)	0.02	0.019 (↓5%)	0.018* (↓10%)	0.023 (↑15%)	0.027	0.027 (0%)	0.022* (↓19%)	0.022 (↓19%)
Recovery phase Day 90(% control)	0.021			0.022 (↑5%)	0.021			0.025 (↑19%)
Relative haematocrit
Treatment phase Day 90 (% to control)	0.48	0.49 (↑2%)	0.49 (↑2%)	0.49 (↑2%)	0.46	0.46 (0%)	0.46 (0%)	0.45 (↓2%)
Recovery phase Day 90(% control)	0.47			0.46 (↓2%)	0.43			0.45 (↑15%)
WBC (g/L)
Treatment phase Day 90 (% to control)	5.89	6.51 (↑11%)	5.85 (↓1%)	6.32 (↑7%)	4.07	3.86 (↓5%)	2.77** (↓32%)	2.78** (↓32%)
Recovery phase Day 90(% control)	6.55			5.6 (↓15%)	2.82			2.69 (↓5%)
Relative basophile counts
Treatment phase Day 90 (% to control)	0.004	0.004 (0%)	0.005 (↑25%)	0.005 (↑25%)	0.003	0.004 (↑33%)	0.003 (0%)	0.004 (↑33%)
Recovery phase Day 90 (% control)	0.003			0.005* (↑67%)	0.004			0.004 (0%)
Absolute basophil counts (g/L)
Treatment phase Day 90 (% to control)	0.02	0.03 (↑50%)	0.03 (↑50%)	0.03 (↑50%)	0.02	0.01 (↓50%)	0.01* (↓50%)	0.01 (↓50%)
Recovery phase Day 90 (% control)	0.02			0.03 (↑50%)	0.01			0.01 (0%)
Relative large unstained cell count
Treatment phase Day 90 (% to control)	0.006	0.006 (0%)	0.005 (↓17%)	0.006 (0%)	0.007	0.006 (↓14%)	0.006 (↓14%)	0.006 (↓14%)
Recovery phase Day 90 (% control)	0.009			0.007* (↓22%)	0.007			0.006 (↓14%)
Absolute large unstained cell count (g/L)
Treatment phase Day 90 (% to control)	0.04	0.04 (0%)	0.03 (↓25%)	0.04 (0%)	0.03	0.02 (↓33%)	0.02 (↓33%)	0.02 (↓33%)
Recovery phase Day 90 (% control)	0.07			0.04 (↓43%)	0.02			0.02 (0%)
Absolute monocyte count (g/L)
Treatment phase Day 90 (% to control)	0.16	0.19 (↑19%)	0.14 (↓12%)	0.19	0.10	0.08 (↓20%)	0.08 (↓20%)	0.07* (↓30%)
Recovery phase Day 90 (% control)	0.23			0.16* (↓30%)	0.09			0.08 (↓11%)
Absolute lymphocyte count (g/L)
Treatment phase Day 90 (% to control)	4.34	4.87 (↑12%)	4.42 (↑2%)	4.54 (↑5%)	3.25	3.09 (↓5%)	2.13** (↓34%)	2.11** (↓35%)
Recovery phase Day 90 (% control)	4.62			3.89 (↓16%)	2.07			1.94 (↓6%)
Platelets (g/L)
Treatment phase Day 90 (% to control)	895	941 (↑5%)	885 (↓1%)	946 (↑6%)	1033	963 (↓7%)	910 (↓12%)	894* (↓13%)
Recovery phase Day 90 (% control)	893			759 (↓15%)	871			916 (↑5%)
statistically significant at * p < 0.05 and ** p < 0.01 significance level

Table 4:    Clinical biochemistry findings following 90 days of treatment in rats

Dose level	Males				Females
mg/m³	0	0.1	0.5	5	0	0.1	0.5	5
Creatinine (µmol/L)
Treatment phase Day 90 (% to control)	25.9	21.9* (↓15%)	22.9 (↓12%)	23.9 (↓8%)	28.3	33.7 (↑19%)	28.7 (↑1%)	30.9 (↑9%)
Recovery phase Day 90(% control)	25.1			25.0 (↓0.4%)	27.6			28.2 (↑2%)
Potassium (mmol/L)
Treatment phase Day 90 (% to control)	4.32	4.83* (↑12%)	4.63 (↑7%)	4.54 (↑5%)	3.51	3.74 (↑7%)	3.94* (↑12%)	3.84 (↑9%)
Recovery phase Day 90(% control)	4.05			4.2 (↑4%)	3.37			3.51 (↑4%)
Phosphorus (mmol/L)
Treatment phase Day 90 (% to control)	1.73	1.92* (↑11%)	1.86 (↑8%)	1.79 (↑3%)	1.34	1.46 (↑9%)	1.40 (↑4%)	1.39 (↑4%)
Recovery phase Day 90(% control)	1.55			1.59 (↑3%)	1.07			1.04 (↓3%)
Protein (g/L)
Treatment phase Day 90 (% to control)	66.79	66.66 (↓0.2%)	66.16 (↓1%)	67.98 (↑2%)	70.27	68.6 (↓2%)	66.65 (↓5%)	69.72 (↓1%)
Recovery phase Day 90 (% control)	67.24			69.03* (↑3%)	72.38			73.95 (↑2%)
Albumin (g/L)
Treatment phase Day 90 (% to control)	44.29	44.43 (↑0.3%)	44.58 (↑0.7%)	44.83 (↑1%)	51.96	49.27 (↓5%)	49.2 (↓5%)	50.47 (↓3%)
Recovery phase Day 90 (% control)	41.90			42.84* (↑2%)	51.69			51.87 (↑0.3%)
Albumin/globulin ratio
Treatment phase Day 90 (% to control)	2.04	2.02 (↓1%)	2.00 (↓2%)	1.98 (↓3%)	2.86	2.59* (↓9%)	2.86 (0%)	2.65 (↓7%)
Recovery phase Day 90 (% control)	1.65			1.64 (↓1%)	2.48			2.42 (↓2%)
statistically significant at * p < 0.05 and ** p < 0.01 significance level

 

Table 5:           Organ weight findings (mean ± SD) following 90 days of treatment in rats

Dose level	Males				Females
mg/m³ air	0	0.1	0.5	5	0	0.1	0.5	5
Spleen weight
Mean abs. weight (g)
Treatment phase Day 90	0.72 ± 0.12	0.74 ± 0.15	0.69 ± 0.10	0.71 ± 0.11	0.43 ± 0.04	0.52* ± 0.11	0.47 ± 0.06	0.47 ± 0.07
(% to control)		(↑3%)	(↓4%)	(↓1%)		(↑21%)	(↑9%)	(↑9%)
Recovery phase Day 90	0.74 ± 0.09			0.82 ± 0.12	0.59 ± 0.08			0.58 ± 0.06
(% to control)				(↑11%)				(↓2%)
Mean spleen to bw ratio (%)
Treatment phase Day 90	0.20 ± 0.03	0.20 ± 0.02	0.19 ± 0.08	0.19 ± 0.03	0.20 ± 0.02	0.25** ± 0.04	0.23 ± 0.02	0.23 ± 0.04
(% to control)		(0%)	(↓5%)	(↓5%)		(↑25%)	(↑15%)	(↑15%)
Recovery phase Day 90	0.16 ± 0.02			0.18 ± 0.03	0.24 ± 0.03			0.24 ± 0.03
(% to control)				(↑13%)				(0%)
Mean spleen to brain weight ratio (%)
Treatment phase Day 90	35.71 ±7.01	35.44 ±6.53	33.29 ±4.86	35.04 ±6.22	22.95 ±	27.26* ± 5.27	25.65 ± 3.32	25.25 ± 3.26
(% to control)		(↓1%)	(↓7%)	(↓2%)		(↑18%)	(↑12%)	(↑10%)
Recovery phase Day 90	34.69 ± 4.06			38.07 ± 4.68	30.35 ± 3.94			30.21 ± 3.72
(% to control)				(↑10%)				(↓0.5%)
Kidney weight
Mean abs. weight (g)
Treatment phase Day 90	2.07 ± 0.16	2.22 ± 0.29	2.16 ± 0.21	2.22 ± 0.29	1.41 ± 0.09	1.44 ± 0.15	1.36 ± 0.08	1.34 ± 0.08
(% to control)		(↑7%)	(↑4%)	(↑7%)		(↑2%)	(↓4%)	(↓5%)
Recovery phase Day 90	2.34 ± 0.22			2.32 ± 0.24	1.68± 0.15			1.52** ± 0.09
(% to control)				(↓1%)				(↓10%)
Mean kidney to bw ratio (%)
Treatment phase Day 90	0.58 ± 0.05	0.60 ± 0.04	0.61 ± 0.06	0.60 ± 0.04	0.66 ± 0.05	0.71 ± 0.06	0.67 ± 0.04	0.66 ± 0.05
(% to control)		(↑3%)	(↑5%)	(↑3%)		(↑8%)	(↑2%)	(0%)
Recovery phase Day 90	0.52 ± 0.05			0.51 ± 0.03	0.69 ± 0.05			0.63* ± 0.05
(% to control)				(↓2%)				(↓9%)
Mean kidney to brain weight ratio (%)
Treatment phase Day 90	102.69 ± 9.09	106.49 ± 10.95	104.16 ± 8.62	108.57 ± 10.58	74.49 ± 3.97	76.30 ± 7.03	74.42 ± 4.15	72.36 ± 4.98
(% to control)		(↑4%)	(↑1%)	(↑6%)		(↑2%)	(0%)	(↓3%)
Recovery phase Day 90	109.38± 10.74			108.21 ± 8.90	86.31 ± 6.70			79.64* ± 4.84
(% to control)				(↓1%)				(↓8%)
Uterus weight
Mean abs. weight (g)
Treatment phase Day 90					1.25 ± 0.39	1.05 ± 0.42	0.86 ± 0.22	1.16 ± 0.42
(% to control)						(↓16%)	(↓31%)	(↓7%)
Recovery phase Day 90					1.64 ± 0.48			1.27 ± 0.20
(% to control)								(↓23%)
Mean uterus to bw ratio (%)
Treatment phase Day 90					0.59 ± 0.20	0.51 ± 0.17	0.42 ± 0.10	0.58 ± 0.23
(% to control)						(↓13%)	(↓29%)	(↓2%)
Recovery phase Day 90					0.67 ± 0.21			0.52 ± 0.08
(% to control)								(↓22%)
Mean uterus to brain weight ratio (%)
Treatment phase Day 90					66.30 ± 19.71	55.51 ± 21.04	47.23 ± 11.72	63.55 ± 25.06
(% to control)						(↓16%)	(↓29%)	(↓4%)
Recovery phase Day 90					83.79 ± 23.47			66.54* ± 9.71
(% to control)								(↓21%)
abs.: absolute; bw: body weight statistically significant at * p < 0.05 or ** p < 0.01 significance level

 

 

Table 6: Incidence and Mean Severity Grade of Main Findings in Lungs

Finding Incidence/Mean severity grade	Control		0.1 mg/m³ air		0.5 mg/m³ air		5 mg/m³ air
Main study (non-recovery)	10 M	10 F	10 M	10 F	10 M	10 F	10 M	10 F
Alveolar macrophages phagocytizing blackish material Incidence/mean severity	0	0	1/1.0	1/1.0	10/1.0	10/1.0	10/2.0	10/2.0
Intra-alveolar conglomeration of macrophages phagocytizing blackish material Incidence/mean severity	0	0	0	0	5/1.0	3/1.0	10/1.0	10/1.0
Deposition of blackish material in the interstitium at bronchiolar-alveolar area Incidence/mean severity	0	0	0	0	0	0	3/1.0	4/1.0
Recovery	10 M	10 F	-	-	-	-	10 M	10 F
Alveolar macrophages phagocytizing blackish material Incidence/mean severity	0	0					10/1.3	10/1.4
Intra-alveolar conglomeration of macrophages phagocytizing blackish material Incidence/mean severity	0	0					10/1.0	10/1.0
Deposition of blackish material in the interstitium at bronchiolar-alveolar area Incidence/mean severity	0	0					3/1.0	3/1.0
Severity grades were generally assigned as GRADE 1 = Minimal / very few / very small GRADE 2 = Slight / few / small GRADE 3 = Moderate / moderate number / moderate size GRADE 5 = Massive / extensive number / extensive size

 

 

Table 7: Summary of Differential Cell Count on BALF Smear: Group Mean Ratio (%) of Each Cell Type - Non-Recovery Males and Females

Group Mean Ratio (%) ± SD	Macrophages					Eosino-phils	Neutro-phils	Lympho-cytes	Other cells	Epithelial cells
	Total	Non-BM laden	BM-laden
			Sum	Not multi-NC	Multi-NC
Non recovery males
Control	96.9 ± 1.16	96.6 ± 1.16	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.04 ± 0.09	0.82 ± 0.74	0.70 ± 0.33	0.06 ± 0.09	1.78 ± 0.67
0.1 mg/m³	97.3 ± 0.87	96.7 ± 1.12	0.60 ± 0.34	0.60 ± 0.34	0.00 ± 0.00	0.04 ± 0.05	0.38 ± 0.04	0.56 ± 0.13	0.04 ± 0.05	1.72 ± 0.69
0.5 mg/m³	96.30 ± 1.73	94.30 ± 1.63	2.10* ± 0.35	2.06 ± 0.33	0.04 ± 0.05	0.04 ± 0.09	0.38 ± 0.26	0.74 ± 0.52	0.08 ± 0.11	2.44 ± 1.04
5 mg/m³	97.30 ± 1.25	87.70* ± 2.29	9.64** ± 2.48	9.22** ± 2.60	0.40** ± 0.24	0.02 ± 0.04	0.44 ± 0.31	0.40 ± 0.02	0.02 ± 0.04	1.84 ± 1.05
Non recovery females
Control	98.60 ± 0.71	98.60 ± 0.71	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.0	0.05 ± 0.07	0.20 ± 0.14	0.40 ± 0.00	0.00± 0.00	0.75 ± 0.64
0.1 mg/m³	97.90 ± 0.77	97.50 ± 0.65	0.38 ± 0.26	0.38 ± 0.26	0.00 ± 0.00	0.00* ± 0.00	0.38 ± 0.26	0.36 ± 0.13	0.04 ± 0.05	1.30 ± 0.68
0.5 mg/m³	96.80 ± 1.19	94.90* ± 1.85	1.86 ± 1.37	1.82 ± 1.35	0.04 ± 0.09	0.00* ± 0.0	0.58 ± 0.63	0.34 ± 0.22	0.04 ± 0.05	2.24 ± 0.58
5 mg/m³	97.5 ± 0.86	88.30** ± 1.92	9.20** ± 1.35	8.36** ± 1.51	0.84 ± 0.62	0.00* ± 0.00	0.28 ± 0.16	0.34 ± 0.11	0.06 ± 0.13	1.78 ± 0.77
Non-BM laden: Macrophages not phagocytizing blackish materials (test item) BM-laden: Macrophages phagocytizing blackish materials Not Multi-Nc.: BM-laden macrophages not showing multinucleated syncytial giant cell appearance Multi-Nc.: BM-laden macrophages showing multinucleated syncytial giant cell appearance. statistically significant at * p < 0.05 or ** p < 0.01 significance level

 

Table 8: Summary of Differential Cell Count on BALF Smear: Group Mean Ratio (%) of Each Cell Type - Recovery Males and Females

Group Mean Ratio (%) ± SD	Macrophages					Eosino-phils	Neutro-phils	Lympho-cytes	Other cells	Epithelial cells
	Total	Non-BM laden	BM-laden
			Sum	Not multi-NC	Multi-NC
Recovery males
Control	98.10 ± 0.83	98.10 ± 0.83	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.22 ± 0.13	0.20 ± 0.10	0.02 ± 0.04	1.46 ± 0.72
5 mg/m³	97.60 ± 0.57	92.20** ± 0.93	5.42** ± 0.60	4.38** ± 0.47	1.04** ± 0.34	0.00 ± 0.00	0.36 ± 0.18	0.28 ± 0.08	0.02 ± 0.04	1.68 ± 0.68
Recovery females
Control	97.50 ± 1.24	97.50 ± 1.24	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.05 ± 0.10	0.40 ± 0.28	0.25 ± 0.06	0.03 ± 0.05	1.79 ± 1.08
5 mg/m³	96.80 ± 1.47	91.20** ± 1.48	5.58** ± 1.43	4.40** ± 1.74	^1.16**± 0.45	0.00 ± 0.00	0.52 ± 0.38	0.34 ± 0.21	0.08 ± 0.11	2.26 ± 1.22
Non-BM laden: Macrophages not phagocytizing blackish materials (test item) BM-laden: Macrophages phagocytizing blackish materials Not Multi-Nc.: BM-laden macrophages not showing multinucleated syncytial giant cell appearance Multi-Nc.: BM-laden macrophages showing multinucleated syncytial giant cell appearance. statistically significant at * p < 0.05 or ** p < 0.01 significance level

Applicant's summary and conclusion

Conclusions:

The study was conducted according to OECD guideline 413 and under GLP conditions.

Groups of Wistar rats were exposed to the test material via inhalation for 13 weeks (5 days per week, 6 h per day via nose-only exposure). The concentrations were 0.05, 0.5 and 5 mg/m³ air, animals of the control group were exposed to air. For the vehicle and high dose group, recovery groups were included in the test and sacrificed 13 weeks after the termination of treatment.

No adverse effects were observed in any of the treatment groups. The only treatment related difference between control and treatment groups was the occurence of macrophages phagocytizing blackish material (i.e., test item) both in the broncho-alveolar lavage fluid (BALF) and lung tissue of all test item-treated groups in a dose-dependent manner. However, the amounts of macrophages in the lung of animals from the test item-treated groups were within the range of that from the controls, and no obvious increase in the number of macrophages were identified in any animals of the test item treatment groups.
There was no further evidence of inflammatory responses or other degenerative and/or proliferative lesions that could be attributable to treatment with the test item. Therefore, due to relative increase in the ratio of blackish material-laden macrophages, the histomorphologic appearances indicated physiological processes for elimination of foreign materials.
In the recovery groups, macrophages phagocytizing blackish material were still present but the ratio (%) to total number of cells counted decreased compared to the ratio obtained in the non-recovery animals. No inflammatory response was observed so that the finding was not considered adverse.

Based on the results of this study, the no-observed-effect-concentration (NOEC) and the no-observed-adverse-effect-concentration (NOAEC) were established at 5.0 mg/m3 air due to the absence of histological indicators of the inflammatory, degenerative and/or proliferative responses that may be elicited by persistence of the test item in the lung as well as any other organs of the test item treated animals.

Generally, larger particles are prone to be trapped or deposited in the nose and upper respiratory tract, and only the smaller size fractions will reach and be deposited in the more peripheral bronchioles and proximal alveolar region. In the present study, the mean MMAD (mass median aerodynamic diameter) in groups 0.5 and 5 mg/m³ was larger than 11 μm and the mean percentage of particles less than 3 μm was lower than 25%. From this, it is likely that the larger test item population which could force the excessive uptake by macrophages was more or less entrapped and excluded in the upper respiratory tract.