Tangled Multi-Walled Carbon Nanotubes

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2015. This study was not proposed in a test plan. This study was performed under a US EPA Toxic Substances Control Act (TSCA) 5(e) consent order.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Inhalation toxicity testing: expert meeting on potential revisions to OECD test guidelines and guidance document

Version / remarks:

Series Safety Manufactured Nanomaterials. 2012;35:[ENV/JM/MONO(2012)14]

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation:
males: ca. 250-290g
females: ca. 160-220 g
- Fasting period before study: no
- Housing: in groups of maximally four in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Pelleted standard Harlan Teklad 2914C rodent main-tenance diet, ad libitum
- Water: Community tap-water, ad libitum
- Acclimation period: At least five days under test conditions after health examination. Only animals without any visible signs of illness will be used for the study. Animals of allocations A to D, Groups 1 to 4 will be accustomed to the restraining tubes during acclima-tization period for 3 daily periods of approximately 1, 2, and 4 hours.

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: 02/2014 to: 05/2015

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Remarks:

flow-past

Vehicle:

clean air

Remarks on MMAD:

The mean mass median aerodynamic diameter (MMAD) (gravimetric determination) was between 1.57 and 2.30 µm, showing that the generated aerosols were within the respirable range for rats.
GSD values were 2.53 and 2.47 at the mid and high concentrations, respectively.
Count median aerodynamic diameter (CMAD, nm) (WPRS determination) were 196.2 ± 54.7, 231.5 ± 65.1 and 208.0 ± 62.0 at the low, mid and high concentrations, respectively.
Mean percentage of particles < 3 µm (gravimetric determination) were 77.4 and 62.1 at the mid and high concentrations, respectively.
At 0.05 mg/m3 due to the very low concentration, the particle size could not be determined by gravimetry at an air flow rate of 1 L/min. The aerosol
concentrations at 0.05 and 0.25 mg/m3 were achieved by serial dilution with compressed, filtered, dry air of the 0.25 and 5.0 mg/m3 concentrations, respectively.
Therefore, the MMAD and GSD at 0.05 mg/m3 are expected to be of the same order as at 0.25 mg/m3. This is also confirmed by the CMAD and the particle size data from the 5-day study with sampling at an air flow rate of 9 L/min.

Particle Size Determination: Wide-range Particle Spectrometer (WPS) (mean values)
Group 2 Group 3 Group 4
CMAD [nm] GSD CMAD [nm] GSD CMAD [nm] GSD
196 3.51 231 2.90 208 2.96

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Preparation of Test Item: Prior to use for aerosol generation, the test item was ground in a ball mill for 12 hours under an Argon atmosphere and sieved at a mesh size of 63 µm, in order to facilitate aerosol generation and an acceptably small particle size (see attached letter report of 27-Apr-2012 and 23-May-2013). The resulting fine particles were used in the study.
- 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 aerosol was discharged constantly through the exposure system and exhausted using a tubing/filter system.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes.
- Source and rate of air: between 0.75 to 1 L/min, which is sufficient to minimize re-breathing of the test aerosol as it is more than twice the respiratory minute volume of a rat.
- System of generating particulates/aerosols: A dust aerosol was generated from the milled test item using a SAG 410 Solid Aerosol Generator (Topas GmbH, Dresden, Germany) connected to a micronizing jet mill and a cyclone and two elutriators thereafter. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer.
- Temperature, humidity, O2 in air chamber:
Group Mean Temperature (°C) Mean Relative Humidity (%) Mean Oxygen Concentration (%)
1 23.2 ± 0.9 5.8 ± 1.5 20.8 ± 0.0
2 23.3 ± 0.7 6.1 ± 1.7 20.8 ± 0.0
3 23.6 ± 0.7 6.1 ± 1.7 20.8 ± 0.0
4 23.8 ± 0.6 6.3 ± 1.6 20.8 ± 0.0
- 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 at least once per week for groups 2 to 4, if feasible. In addition, impactor samples in groups 2 and 3 were collected over several days of exposures when considered to be necessary. The airflow rate through the impactor was approximately 1 L/min.
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA).
In addition, the aerosol was also analyzed by a Wide Range Particle Spectrometer™ (Model 1000XP, MSP Corporation, Shoreview, USA) in the size range of 5 nm to 10 µm at least once per week for groups 2 to 4. The sampling airflow rate was approximately 1 L/min. The MMAD and the GSD was reported.

TEST ATMOSPHERE
- Brief description of analytical method used:
See above.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric determination of the aerosol concentration was performed three to six times during each exposure for group 4. The duration of sampling was sufficient to ensure reliable results. In groups 2 and 3, two filters were sampled in parallel. In group 2, the filter samples were weighed at least 12 hours after the end of sampling in order to have similar humidity conditions as for the weighing before sampling start and the same filters were used for consecutive sampling as appropriate.
In addition, the aerosol was analyzed once per week with a Wide Range Particle Spectrometer™ (WRPS, Model 1000XP, MSP Corporation, Shoreview, USA) in the size range of 5 nm to 10 µm. The sampling airflow rate was approximately 1 L/min. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6-hours daily, 5 days/week at approximately 24-hour intervals

Dose / conc.:

0.05 other: mg/m³ air (target)

Remarks:

Group 2, 0.059 mg/m³ air (analytical)

Dose / conc.:

0.25 other: mg/m³ air (target)

Remarks:

Group 3, 0.279 mg/m³ air (analytical)

Dose / conc.:

5 other: mg/m³ air (target)

Remarks:

Group 4, 4.84 mg/m³ air (analytical)

No. of animals per sex per dose:

35 (see enclosed table 1)

Control animals:

other: yes, sham-exposed (group 1)

Details on study design:

- Dose selection rationale: Concentrations were selected based on data from a 5-day study (Schuler, 2010, study C89486) and the results of 90-day inhalation studies published on analogue carbon nanotubes.
- Post-exposure recovery period in satellite groups: 13 and 52 weeks
- Interim sacrifice: after 4 weeks of exposure

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during treatment and once daily during acclimatization and recovery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Was recorded twice daily before and after expo-sure, once daily on week-ends, and once weekly during acclimatization and recovery. In addition, once weekly careful clinical examination of each animal in a standard arena. Clinical signs was be recorded from allocation A to D only.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly (each individual animal, Allocation A to D only) during the first 4 weeks; if no effects 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
Time schedule: twice weekly (each individual animal, Allocation A to D only) during the first 4 weeks; if no effects weekly thereafter and weekly during acclimatization and recovery.

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 and week 13 of treatment
- Dose groups that were examined: All main study animals (Allocation C), using a direct ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13 weeks of treatment and 13 and 52 weeks of recovery
- Anaesthetic used for blood collection: Yes (isoflurane
- Animals fasted: Yes
- How many animals: all animals of allocations A and B (3-months recovery)
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 13 Weeks of treatment
- Anaesthetic used for blood collection: Yes (isoflurane
- Animals fasted: Yes
- How many animals: all animals of allocations A and B (3-months recovery)
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 13 Weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals of Allocation C were observed for behavior, reflexes, activity, responsiveness, urine or feces, posture and general abnormalities once at the end of the treatment period. Animals of Allocation C were observed for locomotor activity once at the end of the treatment period. 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 was reported.
- Dose groups that were examined: all
- Battery of functions tested: grip strength / motor activity / Landing Foot Splay

OTHER:
- BLOOD PRESSURE
Time schedule for examinations: Blood pressure was measured on 5 animals of allocation C during acclimatization and before and after exposure in week 13 and on week 52 of recovery.
- VAGINAL SMEARS
Vaginal smears were taken for 14 days from all females during treatment weeks 11 and 12 for estrous cycle evaluation.
- SPERM ANALYSIS
Sperm analysis was performed on all males of Allocations A, B and C as soon as feasible after broncho-alveolar lavage sampling. Motility, morphology and spermatid and sperm count.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes (see table 5)

HISTOPATHOLOGY: Yes
- Light microscopy: (see table 5)
- Brightfield optical and hyperspectral microscopy:
CytoViva hyperspectral microscopy was employed to spectrally identify Graphistrength C100 MWCNT in the lung, brain, kidney, and liver tissues of Wistar rats exposed to 5.0 mg/m3 of Graphistrength C100 via inhalation for 13 weeks and after a 52-week treatment-free period. The samples were imaged with transmitted brightfield illumination to allow for pathological examination of the H&E stained tissue samples. Each tissue sample was surveyed visually to identify any dark particles that appeared to be Graphistrength C100.

Other examinations:

BRONCHO-ALVEOLAR LAVAGE FLUID
- How many animals: all animals from allocation A, B, C and D were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination approximately 22 – 26 h after the last 4-week and 13-week exposure and at the end of the 13- and 52-week recovery periods.
- Parameters checked: lactate dehydrogenase, alkaline phosphatase, gamma-glutamyltransferase, phospholipids, total protein, total cell count, cell viability, differential cell count and cytokines TNF-a, IL-1a, IL-1ß and IL-5.

Statistics:

The food consumption, blood pressure, grip strength, landing foot play, body temperature, body weight, food consumption, blood clinical laboratory and BALF parameters, organ weights, and sperm parameters were analysed for statistical significance by the Dunnett-test (many to one t-test) based on a pooled variance estimate, if the variables can 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 for the locomotor activity, urinalysis, and BALF biochemical parameters instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution. Fisher's exact test was applied to the ophthalmoscopy and macroscopic findings. Nonparametric Mann-Whitney test was applied to the cytokines test results as data did not follow a normal distribution.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs in any group that were considered to be related to exposure to the test item. Hair loss, scabs, erythema and localized swelling were recorded in a small number of animals. These signs are commonly seen in animals of this age and strain and are, therefore, considered to be incidental.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Exposure to the test item had no effect on body weight and body weight gain development.
Slightly reduced body weight gain was seen in males and females of group 3 and 4 during several weeks of exposure. However, the body weights of the males and females of groups 3 and 4 remained similar to the control group during the exposure period. Increased body weight gain and body weight in males and females of group 4 were observed during recovery.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No effect on food consumption was observed during the 13-week treatment (exposure) period.
Increased food intake was recorded in males of groups 2 to 4 during the first week of recovery and several weeks thereafter in groups 2 and 4. In addition increased food intake was recorded during the first two weeks of recovery in females of group 4.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes in hematology parameters of rats of groups 2 and 3 at the end of the 13-week exposure period or at the end of the 13-week and 52-week recovery periods.
Exposure-related changes were noted for rats of group 4 and consisted of a shift in the differential white blood cell count characterized by an increase in neutrophil counts (relative and absolute) and a decrease in lymphocyte counts (relative). These changes were noted at the end of the 13-week exposure period and at the end of the 13-week recovery period in both sexes. At the end of the 52-week recovery period the changes were reversed in males of group 4, but not in females of group 4 (see Text Table 1). These effects were considered to be most probably secondary to the inflammatory response observed in the lungs of the exposed animals.
The slight changes of the eosinophil counts in males of groups 3 and 4 (increase), prothrombin times in males of group 4 and females of group 3 (shortened), platelets in males of group 4 (increased) observed after the 13-week exposure as well as the slightly shortened prothrombin time in males after 52 weeks of recovery were considered to be fortuitous and to lie within the normal range of biological variation. The values remained within the ranges of historical control data and/or were not dose-related and/or were observed only in one sex and/or occurred at inconsistent time points.
This holds true also for the few isolated additional changes which achieved statistical significance from controls (lower hemoglobin concentration in males of group 2 after 52 weeks recovery; lower hematocrit in males of group 4 after 13 weeks of recovery, in males of group 2 after 52 weeks recovery, in females of groups 2 and 3 after 52 weeks of recovery; higher mean corpuscular hemoglobin concentration in females of group 2 after 52 weeks recovery; decreased high fluorescence ratio in the reticulocyte count in males of group 3 after 13 weeks of exposure and in females of group 4 after 13 weeks recovery).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased potassium values were recorded in males of group 4 (9%) and in all treated groups of females (15%, 22% and 11% in groups 2, 3 and 4, respectively) at the end of the exposure period (see Text Table 2), but not at the end of the 13-week and 52-week recovery periods. Considering the low magnitude of this hyperkalemia, the lack of concentration exposure relationship in females and the variability of potassium levels in rats [see References (5)], these changes were not considered to be exposure related and/or adverse even though the results were partially out of the range of historical control data.
The few other statistically significant differences from controls noted were considered to be fortuitous and not related to exposure to the test item. The values remained within the ranges of historical control data and/or were not dose-related and/or were observed only in one sex and/or occurred at inconsistent time points or were due to an unusual control value.
These changes consisted of an increase of glucose concentration in males of group 3 after 52 weeks recovery, a decrease of creatinine concentration in males of groups 2 and 3 after the 13-week recovery and females of group 4 after the 13-week exposure, a decrease of triglyceride concentration in males of groups 2 and 3 after the 13-week exposure and in females of group 3 and 4 after 13 weeks recovery, an increase in the phospholipid concentration in males of group 4 after the 52-week recovery, a decrease in lactate dehydrogenase activity in males of group 4 after
13-week exposure and in males of groups 2 and 3 after 52-week recovery, a decrease of creatine kinase activity in females of group 3 after 13-week recovery, an increase of sodium concentration in females of group 4 after the 13-week exposure and in females of group 2 after 13-week recovery, an increase of chloride concentration in females of group 4 after the 13-week exposure, a decrease of calcium concentration females of group 3 after 13-week recovery and a decrease of protein concentration in females of groups 4 after 13-week exposure and after 13-week recovery as well as an increase of protein concentration in females of group 4 after 52-week recovery.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on urinalysis parameters.
The few statistically significant differences from controls noted were consider to be chance findings and to lie within the range of biological variation.
These changes were isolated, not dose-related, occurred in one sex, only, and were within the range of the historical control data. They consisted of an increased urine volume in females of group 3 after 13-week recovery, an increase of the urine pH in females of group 2 after 13-week exposure and an increased number of leukocytes in males of group 3 after 13-week exposure.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

A small number of typical background changes were noted during detailed weekly clinical observations. All were considered to be unrelated to the exposure to the test item.
During the treatment and recovery periods, findings consisted mostly of localized hair loss or small dermal injuries, and other dermal findings. Two females (nos. 187 and 204) of group 2 had minor subdermal swelling; such findings were not seen at higher dosages and therefore considered to be unrelated to the exposure to the test item.

Functional Observational Battery
There were no abnormal findings noted during functional observational battery.

Grip Strength
There were no effects on grip strength that were considered to be related to the exposure to the test item.

Locomotor Activity
There were no effects on locomotor activity that were considered to be related to the exposure to the test item.

Landing Foot Splay
There was no effect on the landing foot splay in any group exposed to the test item.
The group average values recorded for controls and exposed groups were distances between 5.8 cm to 7.4 cm for males and 3.8 cm to 5.3 cm for females.

Body Temperature
There was no effect on body temperature in any group exposed to the test item.
The group average values recorded for controls and exposed groups were in the range of 36.6 °C to 36.9 °C for males and between 37.1 °C to 37.5 °C for females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Exposure to the test item had no effect on organ weights of male and female rats in groups 2 and 3.
Increased absolute and relative weights of the lungs were recorded in rats of group 4 on all occasions, i.e. after 4 and 13 weeks of exposure and after the 13-week and 52-week recovery periods (see Text Table 3).
After 4 weeks of exposure, incidental decreases were observed in the absolute liver weight in males of group 2, in the absolute and relative (to brain) weights of the thymus in males of group 3 and in the absolute and relative (to brain) weights of the spleen in males of groups 2 and 4. All these changes were related to the lower terminal body weights and were no longer observed after 13 weeks of exposure. In females of group 4 absolute organ weights for heart, kidneys and liver as well as the organ weights relative to brain weight for heart and kidneys were increased. These changes are considered to be due to statistically higher body weight in the high dose group and not an effect of the test item (see Text Table 3).
After 13 weeks of exposure, heart weight relative to body weight was increased in females of group 2 and 4 due to a lower terminal body weight of these groups compared to controls. An incidentally higher relative (to body weight) weight of the liver was observed in females of group 3 (see Text Table 3).
In addition after 13 weeks and after 52 weeks of recovery increased liver weights (absolute and relative to brain weight) were recorded in males of group 4 due to a higher terminal body weight compared to controls. Furthermore after 13 weeks of recovery, an incidentally higher relative (to brain) weight of the kidneys was observed in males of the group 3. Brain and heart weight relative to body weight were increased in females of this group but this was considered to be due to lower terminal body weight. Slightly increased lung and heart to body weight ratio in group 3 was considered to be secondary to the lower terminal body weight as well. An incidentally higher relative (to body weight) weight of the thymus was observed in females of group 3 (see Text Table 3).
Few additional statistically significant differences between test item-treated groups and control group 1 were observed after 52 weeks of recovery. These did not show a clear dose-relation, were contrary between the two sexes and/or had not been present after exposure and were therefore considered to be incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no necropsy findings which were considered to be related to exposure to the test item in rats of groups 2 and 3.
Dark red discoloration of the lung was recorded in all males and females of group 4 after 4 weeks of exposure. Black brown foci in the lung and black brown discoloration of the bronchial lymph nodes were recorded in all or most animals of group 4 after 13 weeks of exposure.
Black brown discoloration of the lung and/or greenish foci was seen in most of the animals of group 4 after the 13-week recovery period. Black discoloration was also recorded in the bronchial lymph nodes of most animals and in the mandibular lymph node of one female of this group. One male of group 4 had nodules in the epididymal region (most likely to be a nodule of fat necrosis which is seen occasionally particularly in that area or less likely a sperm granuloma).
Black discoloration of the lungs and tan foci in the lungs were recorded for all males and females of group 4 at the end of the 52-week recovery period. In addition, black discoloration of the bronchial lymph nodes was noted for each 7/10 group 4 males and females. Male no. 129 of group 4 had a pale mass (30x15 mm), part firm/part soft in the abdominal cavity (diagnosed as nodular fat necrosis at microscopic examination).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Light microscopy
Lungs
A concentration-related deposition of variably-sized and shaped black particles, localized within the alveolar macrophages when the deposition was minimal (group 2) or slight (group 3) and additionally within tissue macrophages or free within the alveolar lumen when the deposition was moderate or marked (group 4), was recorded in most animals at all sacrifice periods (see Text Table 4). The severity of the deposition increased in animals of group 4 between the 4th and 13th week of exposure. Black particles were also occasionally observed in the sub-epithelial area of the bronchial bifurcations, within macrophages or free in the tissue. When compared to the 13-week exposure period, black particle deposition in the lungs decreased at the end of the 13-week recovery period for groups 2 and 3 while in group 4 it was overall similar at both time periods. When compared to the 13-week recovery period, black particle deposition was overall similar in the lungs at all concentrations at the end of the 52-week recovery period.
In groups 3 and 4, the black particle deposition was always associated to a minimal to moderate presence of alveolar macrophages related to a continuous clearance process. Alveolar macrophages were characterized by intra-alveolar aggregation of enlarged cells with foamy cytoplasm containing, for many of them, black particles.
In group 4, a minimal to moderate intra-alveolar eosinophilic material deposition, considered to be the result of macrophages membrane cell rupture, associated with a minimal alveolar granulocyte infiltration, and a minimal to slight interstitial inflammation and bronchiolar cell hypertrophy/hyperplasia were observed after 13 weeks of exposure and 13 and 52 weeks of recovery with a similar intensity. The interstitial inflammation was mainly seen around the alveolar ducts, at the bronchiole-alveolar junction and the cell hypertrophy/hyperplasia observed in the terminal and respiratory bronchioles was most likely reactive changes to the surrounding inflammatory process.
An increased number of lymphocytes was observed in the bronchus associated lymphoid tissue (BALT) of some males and females of group 4 at the 4 and 13 weeks exposure necropsies (see Text Table 9).
After 4 weeks of exposure, the dark red discoloration observed at necropsy in the lungs of all males and females of group 4 correlated histologically with the deposition of black particles and alveolar macrophages while after 13 weeks of exposure, black particle deposition, alveolar macrophages and/or alveolar eosinophilic material were the histological correlates of the black brown lung foci recorded at necropsy.
Minimal to slight focal/multifocal alveolar septae fibrosis, in areas of intra-alveolar eosinophilic material deposition, was recorded in a few animals of group 4 at the end of the 13- and 52-week recovery periods. Minimal to slight focal/multifocal granulomatous fibrosing inflammation was also seen in 2/10 females of group 4 at the end of the 52-week recovery period. These changes were considered to be a local irritating reaction to the eosinophilic material. Minimal deposition of black particles was seen at the tracheal bifurcation in some animals of group 4 after 13 weeks of exposure and 13 and 52 weeks of recovery but was not associated with inflammatory tissue reaction.

Tracheobronchial Lymph Nodes
Minimal to moderate concentration-related deposition of black particles in the tracheobronchial lymph nodes probably consistent with continuous drainage of black particles from the lungs, was recorded in group 3 and 4 animals after 13 weeks of exposure and 13 and 52 weeks of recovery (see Text Table 5).It was associated with a reversible minimal to moderate in group 4 and a minimal in group 3 increase of lymphocytes within the cortex/paracortex and a minimal to slight reversible vacuolation of the endothelial cells lining the high endothelial venules.

Nasal Cavity and Larynx
Increased incidence/severity of eosinophilic globules were seen in the respiratory and olfactory epithelium of the nasal cavity of group 4.
Cytoplasmic eosinophilic globules (inclusions) in the respiratory and olfactory epithelial cells were observed with increased incidence/severity in males and females of group 4 after 13 weeks of exposure and 13 and 52 weeks of recovery (see Text Table 6). Such findings are frequently observed in inhalation studies (Renne et al., 2009 ) and are considered to be evidences of irritating effects (Greaves, 2007 ).
Minimal squamous metaplasia was observed in the larynx of 2/10 males of group 3 and 4/10 males and 5/10 females of group 4 at the end of the 13 week exposure. The finding present the ventral glands at the base of the epiglottis is a common reaction to inhaled material and, when of minimal severity, is considered to be a non-adverse adaptative change (Kaufmann et al., 2009 ). At the end of the 13-week recovery period, minimal squamous metaplasia was also recorded in the larynx of 1/10 males of group 4; this finding is also known to occur in control animals (Kaufmann et al., 2009) and owing to its single occurrence and minimal severity, a relationship to the test item was considered to be unlikely.

Renne R, Brix A, Harkema J et al. Proliferative and non-proliferative lesions of the rat and mouse respiratory tract. Toxicol Pathol. 37(7 Suppl):5S-73S, 2009
Greaves P. Respiratory tract. In: Histopathology of Preclinical Toxicity Studies, 3rd edition. Academic Press. p223, 2007.
Kaufmann W, Bader R, Ernst H et al. 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. 61:591-603, 2009

Right Testis / Right Epididymis
Seminiferous tubule atrophy/degeneration was observed in the right testis of control and treated rats with a relatively high incidence (see Text Table 7). Although higher severity grade was recorded in a few treated rats, the mean severity was overall similar at 0 and 5.0 mg/m3 air. Seminiferous tubule atrophy/degeneration was the histological correlate of the testes found to be reduced in size at necropsy. The seminiferous tubule atrophy/degeneration, when moderate or severe, was associated with oligospermia in the ipsilateral epididymis. Abnormal content (increased number of intraluminal exfoliated immature germ cells) was also occasionally observed, associated with minimal or slight seminiferous tubule atrophy/degeneration.
These effects were most likely related to the stress associated with immobilization in the restrainer during the inhalation procedure (Lee et al. 1993 ). The higher severity grade observed in a few rats at 5.0 mg/m3 air (associated with oligospermia in the epididymis) was considered to be fortuitous.
Lee et al. Testicular degeneration and spermatid retention in young male rats. Toxicol Pathol. 21(3): 292-302 1993
All other findings were those commonly seen as spontaneous changes in the rat and bore no relationship to the test item. Specifically, there was no test-item finding on the pleura, heart, aorta and olfactory bulb.

- Brightfield optical and hyperspectral microscopy
In most of the lung samples of the exposed animals, the black agglomerates were identified as Graphistrength C100 with high fidelity. There was no Graphistrength C100 translocation in any of the distal organ tissues (brain, kidney and liver).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Analysis of Broncho-Alveolar Lavage Fluid
Cell Count and Viability
There were no test item-related effects in rats of group 2 at any time point.
Exposure-related changes were primarily noted in rats of group 4 at all time points and occasionally in rats of group 3. At the end of the 52-week recovery period, the changes were completely reversed in rats of group 3, but still present in rats of group 4. When present in rats of groups 3 and 4 at the same time, the changes were dose-related (see Text Table 8).
The following statistically significant differences from controls were recorded and attributed to exposure to the test item:
- An increased cell count in males of group 4 after 4 weeks of exposure, in females of group 4 after 13 weeks of exposure, in males of group 3 and both sexes of group 4 after the 13-week recovery and in females of group 4 after the 52-week recovery.
- A decreased percentage of macrophages in both sexes of group 4 on all occasions and in both sexes of group 3 after 13 weeks of exposure.
- An increased percentage of lymphocytes in both sexes of group 4 on all occasions and in males of group 3 after 13 weeks of exposure.
- An increased percentage of neutrophils in both sexes of group 4 on all occasions and in both sexes of group 3 after 13 weeks of exposure.
The slightly reduced percentage of viable cells recorded for males of groups 2 and 4 at the end of the 52-week recovery, were considered to lie within the normal range of biological variation, although they achieved statistical significance. There was no clear dose-relationship, the changes were slight (-7% less than in controls for group 2, -12% less than controls in group 4), occurred in one sex only and were only noted at the end of the 52-week recovery. The only other statistically significant difference from controls was noted in the percentage of eosinophils in males of group 3 after 13-week recovery: 0.1% versus 0.0% versus controls. In the absence of an effect at the high dose and in females, this change was considered to be a chance finding.

Clinical Laboratory Parameters
There were no test item-related effects in rats of group 2 at any time point.
Exposure-related changes were predominantly noted in rats of group 4 at all time points and occasionally in rats of group 3. At the end of the 52-week recovery period, the changes were reversed in rats of group 3, but still present in rats of group 4. When present in rats of groups 3 and 4 at the same time, the changes were dose-related (see Text Table 9).
The following, generally statistically significant differences from controls were recorded and attributed to exposure to the test item:
- An increased concentration of phospholipids in both sexes of group 4 on all occasions (after 4 weeks of exposure, after 13 weeks of exposure, after 13-week recovery and after 52-week recovery) as well as in males of group 3 after 13 weeks of exposure.
- An increased activity of lactate dehydrogenase in both sexes of group 4 at all time points.
- An increased activity of alkaline phosphatase in both sexes of group 4 at all time points.
- An increased activity of gamma glutamyl transferase in both sexes of group 4 at all time points and in both sexes of group 3 after 13 weeks of exposure. Although the difference from controls (+120%) in males of group 4 after 4 weeks of exposure did not achieve statistical significance it is considered to be test item related.
- An increased protein concentration in both sexes of group 4 after 4 week and 13 weeks of exposure and after the 13-week and 52-week recovery periods. Although the difference from controls (+241%) in males of group 4 after 4 weeks of exposure did not achieve statistical significance it is considered to be test item related.
The only other statistically significant difference from controls noted was a slightly increased gamma glutamyl transferase activity (+61% compared with controls) in males of group 3 after the 52-week recovery period. As there was no effect noted in this group after the 13-week recovery or in females after 13 and 52-week recovery and in view of the small extent this difference is considered to be a chance finding and to lie within the normal range of biological variation.

Cytokine Measurements
Allocations A, B and D:
In addition to the statistical significance, cytokine levels which were higher than the Mean + 2 x SD (standard deviation) of unstimulated controls (group 1) were concluded to be biologically significantly increased.
There was no effect on the concentration of cytokines in rats of group 2 at any time point.
A statistically and biologically significant increase of IL-1ß levels was observed in females of group 4 after 13 weeks of exposure. A statistically or biologically significant increase in IL-1ß levels was noted in females of group 4 after 4 weeks of exposure, in females of group 3 after 13 weeks of exposure and in both sexes of group 4 after 13 weeks of recovery. Since the increases observed after the 13-week recovery period were not biologically significant and in males no corresponding findings were observed at the end of the exposure periods, these increases were considered not to be test item-related.
Additionally, the TNF-a levels in both genders of group 4 after 4 weeks of exposure, groups 3 and 4 after 13 weeks of exposure and groups 3 and 4 after 13 weeks of recovery were statistically and biologically significantly increased (see Text Table 10).
Changes (increases or decreases) in levels of IL-5 and IL-1a occasionally even resulting in (biologically) significant effects were measured in single animals (including controls) at different sampling times without any dose-response relationship, and were therefore considered not to be test item-related.
Allocation C:
After 52 weeks of recovery, TNF-a, IL-1a, IL-1ß and IL-5 levels were below the limit of quantification (LoQ) (16.6, 189 and 169 pg/mL, respectively) for all control and treated groups. except for
IL-1a measurements were in excess of the LoQ (13.7 pg/mL) in 9/10 males and 7/10 females of group 4. Thus, mean IL-1a concentrations were increased for males and females of group 4 after the 52-week recovery (see Text Table 5) when compared with the controls (all values below the LoQ).

Details on results:

OESTRUS CYCLE
There was no difference in estrus cycle between treated and control groups.

SPERM ANALYSIS
Sperm count and motility
There was no effect of the exposure on the sperm counts and motility of sperms at the end of the 13-week exposure and the 13- or 52-week recovery periods (see Text Table 6).
The statistically significant increase in sperm count noted in rats of groups 2 and 3 at the end of the 13-week exposure is considered to be a chance finding in the absence of an effect in rats of group 4.
Percentages of progressive motile and stationary motile sperms were outside the range of the historical control data (from oral and dietary studies) in all groups (control and treated) after the 13-week exposure and 13-week recovery periods. However, there is no statistically significant difference between the control and the treated groups, with the exception of the 13-week recovery period in group 4. Nevertheless, in the absence of histological correlates in the testis and exposure-related effects at the end of the 13-week exposure period, this slight shift (percentage of progressive sperms decreased by 14%, percentage of stationary sperms increased by 28%) was considered to be fortuitous. A statistically significant increase of percentage of non-motile sperms was observed in group 3 after 13 weeks of exposure. In the absence of dose-response relationship, histological correlates in the testis, similar change after recovery and with a value inside the historical control data range, this effect was also considered to be fortuitous.

Morphology
There was no test item-related effect on the morphology of sperms in any group.
There was no statistically significant change in sperm morphology at the end of 13 weeks of exposure and after the 13-week and 52-week recoveries (Text Table 12). Compared to the historical control data, all of the mean values for C and E morphology parameters at the end of 13 weeks of exposure were outside the range, including controls. In the absence of a test-item related effect on the histology of the testis, these changes were considered rather to be due to the exposure-procedure than to the test item. This is supported by the histology findings in the right testis after 13 weeks of exposure.

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

4.84 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No direct systemic toxicity (changes in haematological parameters were secondary to the lungs inflammation)

Key result

Dose descriptor:

NOAEC

Remarks:

local effect (pulmonary inflammation)

Effect level:

0.279 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4.84 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

alveolar duct

alveoli

larynx

lungs

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Text Table1    Hematology

 

		WBC		NEUT		EOS		LYMPH		PT		PLATELETS
		G/L		rel. 1 (G/L)		rel. 1 (G/L)		rel. 1 (G/L)		rel. 1 (sec)		G/L
MALE
Historical Control Data§		3.69 -8.48		0.142 - 0.343 (0.78 - 2.17)		0.011 - 0.031 (0.06 - 0.17)		0.595 - 0.809 (2.28 - 6.26)		0.77 - 1.04 (19.8 - 24.1)		752 - 1248
After 13 Weeks of Exposure
Group 1		6.43		0.197 (1.28)		0.009 (0.06)		0.742 (4.78)		0.73 (23.4)		879
Group 2		7.01		0.220 (1.79)		0.013 (0.10)		0.716 (4.79)		0.73 (23.4)		973
Group 3		7.30		0.211 (1.71)		0.013* (0.10)		0.727 (5.15)		0.76 (22.8)		972
Group 4		8.13		0.327** (2.63**)		0.015* (0.12*)		0.608** (5.05)		0.77* (22.5)		1003*
After 13 Weeks of Recovery
Group 1		7.07		0.237 (1.79)		0.012 (0.09)		0.707 (4.88)		0.85 (23.7)		903
Group 2		7.47		0.223 (1.62)		0.014 (0.11)		0.716 (5.44)		0.86 (23.4)		914
Group 3		7.14		0.283 (2.23)		0.015 (0.11)		0.664 (4.55)		0.85 (23.7)		822
Group 4		8.23		0.374* (2.96**)		0.015 (0.12)		0.577*(4.84)		0.89 (22.8)		901
After 52 Weeks of Recovery
Group 1		5.84		0.295 (1.88)		0.015 (0.08)		0.636 (3.58)		0.82 (23.7)		940
Group 2		5.84		0.295 (1.98)		0.017 (0.10)		0.617 (3.45)		0.79 (24.2)		975
Group 3		5.66		0.325 (1.93)		0.019 (0.10)		0.611 (3.39)		0.81 (23.7)		1011
Group 4		6.69		0.401 (2.65)		0.015 (0.10)		0.539 (3.62)		0.85* (23.1**)		1054

 

	WBC	NEUT	EOS	LYMPH	PT	PLATELETS
	G/L	rel. 1 (G/L)	rel. 1 (G/L)	rel. 1 (G/L)	rel. 1 (sec)	G/L
FEMALE
Historical Control Data§	1.90 -5.82	0.124 - 0.338 (0.38 -1.58)	0.010 - 0.049 (0.03 - 0.14)	0.568 - 0.830 (1.24 - 4.41)	0.77 -1.00 (19.7 - 24.8)	809 - 1308
After 13 Weeks of Exposure
Group 1	4.07	0.146 (0.61)	0.011 (0.04)	0.809 (3.29)	0.73 (23.5)	1144
Group 2	4.05	0.152 (0.61)	0.009 (0.04)	0.808 (3.27)	0.75 (23.0)	1087
Group 3	3.69	0.170 (0.62)	0.016 (0.06)	0.779 (2.87)	0.79** (22.2**)	1042
Group 4	4.96	0.261** (1.21**)	0.012 (0.06)	0.698** (3.54)	0.77 (22.6)	1099
After 13 Weeks of Recovery
Group 1	4.17	0.175 (0.67)	0.017 (0.06)	0.772 (3.27)	0.87 (23.2)	883
Group 2	3.75	0.220 (0.84)	0.019 (0.07)	0.729 (2.71)	0.88 (22.8)	933
Group 3	3.32	0.238* (0.74)	0.018 (0.05)	0.714 (2.40)	0.86 (23.5)	841
Group 4	4.90	0.382** (1.81**)	0.013 (0.07)	0.564** (2.85)	0.89 (22.7)	914
After 52 Weeks of Recovery
Group 1	2.95	0.310 (0.85)	0.019 (0.06)	0.623 (1.90)	0.81 (23.7)	849
Group 2	3.45	0.294 (1.11)	0.015 (0.05)	0.643 (2.12)	0.85 (22.9)	940
Group 3	3.28	0.273 (0.90)	0.014(0.05)	0.660 (2.18)	0.85 (23.0)	939
Group 4	3.12	0.394** (1.28*)	0.021 (0.08)	0.531* (1.63)	0.85 (23.0)	841

 

*/**     Significant at 5% (*), 1% (**)

§      95% tolerance limits. Changes statistically significant and outside the historical control data are in bold type

 

Text Table2    Blood Biochemistry

 

	CREAT.	TRIGLY.		SODIUM	POTASSIUM	CHLORIDE	CALCIUM	PROT.
	µmol/L	mmol/L		mmol/L	mmol/L	mmol/L	mmol/L	g/L
MALE
Historical Control Data	20.7 - 31.4		0.26 - 0.93	143.1 - 149.0	3.43 - 4.44	100.7 - 107.3	2.59 - 2.87	63.52 - 76.48
After 13 Weeks of Exposure
Group 1	23.7		0.48	142.8	4.13	102.3	2.68	66.15
Group 2	24.6		0.38*	142.9	4.20	102.1	2.67	65.39
Group 3	23.6		0.37*	143.9	4.25	103.0	2.69	67.02
Group 4	22.4		0.41	144.2	4.52**	103.0	2.69	66.97

After 13 Weeks of Recovery
Group 1	27.9		0.77	145.5	4.52	103.4	2.75	69.08
Group 2	25.5*		0.68	145.7	4.59	103.2	2.77	68.42
Group 3	24.4**		0.75	145.9	4.58	104.4	2.71	67.41
Group 4	26.3		0.79	145.8	4.73	104.5	2.74	68.24
After 52 Weeks of Recovery
Group 1	29.3		0.99	146.4	4.39	102.7	2.75	71.36
Group 2	27.9		1.28	146.0	4.53	103.2	2.75	70.73
Group 3	28.8		1.36	147.1	4.54	104.3	2.73	69.73
Group 4	28.8		1.59	147.7	4.39	103.2	2.82	72.39

 

	CREAT.	TRIGLY.		SODIUM	POTASSIUM	CHLORIDE	CALCIUM	PROT.
	µmol/L	mmol/L		mmol/L	mmol/L	mmol/L	mmol/L	g/L
FEMALE
Historical Control Data	23.6 - 36.2		0.22 - 0.55	141.4 - 149.0	2.78 - 3.87	100.5 - 108.4	2.57 - 2.90	66.15 - 81.76
After 13 Weeks of Exposure
Group 1	30.4		0.32	143.5	3.43	102.0	2.75	72.27
Group 2	27.7		0.31	144.8	3.95**	103.8	2.76	71.50
Group 3	28.3		0.35	144.0	4.17**	103.4	2.80	72.87
Group 4	27.3*		0.30	145.8**	3.81*	105.3**	2.72	69.27*
After 13 Weeks of Recovery
Group 1	28.8		0.72	143.5	3.40	101.7	2.78	77.11
Group 2	31.9		0.60	146.4**	3.41	104.1	2.76	74.74
Group 3	30.1		0.54*	144.9	3.21	101.9	2.72*	74.78
Group 4	30.6		0.47**	145.1	3.54	104.0	2.74	72.29**
After 52 Weeks of Recovery
Group 1	28.8		0.54	144.6	3.86	102.6	2.67	70.58
Group 2	28.8		0.60	144.2	4.26	102.4	2.67	71.47
Group 3	28.7		0.91	144.7	3.87	102.5	2.69	73.75
Group 4	27.3		0.82	145.3	4.12	103.3	2.74	73.90

*/**Significant at 5% (*), 1% (**)

§      95% tolerance limits. Changes statistically significant and outside the historical control data are in bold type

Text Table 3    Organ Weights

 

			Group1 AirControl	Group 2 0.05 mg/m^3air	Group 3 0.25 mg/m^3air	Group 4 5.0 mg/m^3air
After 4 Weeks of Exposure
MALES		n	5	5	5	5
BODY W. (g)		Mean± SD	341.8±17.6	317.4±14.9	323.5±13.3	323.5±20.6
BRAIN absolute (g)		Mean± SD	2.01±0.08	2.00±0.07	2.02±0.04	2.00±0.10
LUNGS absolute (g)		Mean± SD	1.31±0.16	1.35±0.15	1.44±0.18	1.70**±0.18
LUNGS % body weight		Mean± SD	0.38±0.07	0.43±0.05	0.44±0.05	0.53**±0.07
LUNGS % brain weight		Mean± SD	64.97±7.61	67.31±6.15	71.23±9.94	85.14**±9.59
LIVER absolute (g)		Mean± SD	11.47±0.76	9.99*±0.33	10.78±1.17	10.75±1.01
THYMUS absolute (g)		Mean± SD	0.536±0.044	0.416±0.146	0.362*±0.091	0.442±0.077
THYMUS % brain weight		Mean± SD	26.735±3.285	20.834±7.342	17.857*±4.279	22.151±4.101
SPLEEN absolute (g)		Mean± SD	0.77±0.08	0.67*±0.05	0.69±0.06	0.65*±0.03
SPLEEN % brain weight		Mean± SD	38.17±2.81	33.76±3.02	34.00±3.09	32.72*±2.16
FEMALES		n	5	5	5	5
BODY W. (g)		Mean± SD	185.2±15.2	207.0±13.9	192.6±10.7	213.0*±16.8
BRAIN absolute (g)		Mean± SD	1.82±0.12	1.92±0.10	1.85±0.08	1.92±0.08
HEART absolute (g)		Mean± SD	0.61±0.03	0.69±0.05	0.63±0.06	0.75**±0.07
HEART % brain weight		Mean± SD	33.48±2.12	36.20±1.66	34.27±2.71	39.23**±3.32
	LIVER absolute (g)	Mean± SD	6.56±0.67	7.52±0.78	7.00±0.98	7.84*±0.46
LUNGS absolute (g)		Mean± SD	1.02±0.13	1.05±0.14	1.08±0.15	1.38**±0.14
LUNGS % body weight		Mean± SD	0.55±0.04	0.51±0.05	0.56±0.07	0.65*±0.02
LUNGS % brain weight		Mean± SD	55.91±7.14	54.85±5.99	58.60±9.95	71.97**±5.87
KIDNEYS absolute (g)		Mean± SD	1.23±0.10	1.38±0.14	1.30±0.07	1.55**±0.13
KIDNEYS % brain weight		Mean± SD	67.78±6.78	71.69±4.36	70.27±2.34	80.91**±6.65
			Group1 AirControl	Group 2 0.05 mg/m^3air	Group 3 0.25 mg/m^3air	Group 4 5.0 mg/m^3air
	After 13 Weeks of Exposure
	MALES	n	10	10	10	10
	BODY W. (g)	Mean± SD	392.1±20.8	384.0±24.3	396.0±43.6	387.5±32.2
	BRAIN absolute (g)	Mean± SD	2.20±0.07	2.15±0.13	2.15±0.09	2.13±0.07
	LUNGS absolute (g)	Mean± SD	1.50±0.06	1.48±0.14	1.63±0.13	2.21**±0.29
	LUNGS % body weight	Mean± SD	0.38±0.03	0.39±0.02	0.41±0.02	0.57**±0.05
	LUNGS % brain weight	Mean± SD	68.42±2.80	68.86±5.72	75.81±4.23	103.85**±11.39
	FEMALES	n	10	10	10	10
	BODY W. (g)	Mean± SD	248.9±16.1	229.9±16.2	249.4±23.9	239.6±25.7
	BRAIN absolute (g)	Mean± SD	2.04±0.15	1.97±0.10	2.03±0.09	1.99±0.08
	HEART absolute (g)	Mean± SD	0.77±0.06	0.77±0.06	0.81±0.07	0.80±0.08
	HEART % body weight	Mean± SD	0.31±0.02	0.34**±0.02	0.33±0.02	0.34**±0.02
	LUNGS absolute (g)	Mean± SD	1.19±0.10	1.05*±0.07	1.26±0.09	1.73**±0.18
	LUNGS % body weight	Mean± SD	0.48±0.04	0.46±0.03	0.51±0.04	0.72**±0.04
	LUNGS % brain weight	Mean± SD	58.58±4.49	53.33±3.86	62.09±4.84	86.72**±6.25
	LIVER % body weight	Mean± SD	3.51±0.18	3.64±0.20	3.72*±0.19	3.60±0.14
			Group 1 Air Control	Group 2 0.05 mg/m^3air	Group 3 0.25 mg/m^3air	Group 4 5.0 mg/m^3air
	After 13 Weeks of Recovery
	MALES	n	10	10	10	10
	BODY W. (g)	Mean± SD	488.5±39.1	499.2±43.6	511.7±52.6	525.1±34.4
	BRAIN absolute (g)	Mean± SD	2.29±0.07	2.27±0.11	2.22±0.07	2.27±0.09
	LUNGS absolute (g)	Mean± SD	1.49±0.08	1.58±0.16	1.56±0.07	2.42**±0.30
	LUNGS % body weight	Mean± SD	0.30±0.02	0.32±0.02	0.31±0.03	0.46**±0.05
	LUNGS % brain weight	Mean± SD	65.14±3.18	70.11±8.48	70.40±2.18	106.75**±12.15
	LIVER absolute (g)	Mean± SD	13.63±1.44	14.59±1.40	14.67±1.57	15.51*±1.56
	LIVER % body weight	Mean± SD	2.80±0.24	2.93±0.22	2.87±0.24	2.95±0.14
	LIVER % brain weight	Mean± SD	594.84±65.41	645.23±66.10	661.63±67.45	684.58*±76.27
	KIDNEYS % brain weight	Mean± SD	99.83±9.77	109.01±7.87	111.98*±4.22	108.95±12.95
	FEMALES	n	10	10	10	10
	BODY W. (g)	Mean± SD	314.1±30.9	288.4±32.5	291.0±41.8	270.7*±25.7
	BRAIN absolute (g)	Mean± SD	2.05±0.13	2.07 ±0.11	2.09 ±0.09	2.05±0.12
	BRAIN % body weight	Mean± SD	0.66±0.06	0.73±0.08	0.73±0.07	0.76**±0.08
	HEART % body weight	Mean± SD	0.27±0.02	0.29±0.01	0.31*±0.03	0.32**±0.04
	LUNGS absolute (g)	Mean± SD	1.18±0.14	1.20±0.09	1.27±0.14	1.73**±0.12
	LUNGS % body weight	Mean± SD	0.38±0.04	0.42±0.03	0.44**±0.05	0.64**±0.06
	LUNGS % brain weight	Mean± SD	57.76±5.47	58.40±5.36	60.60±4.97	84.86**±8.94
	THYMUS % body weight	Mean± SD	0.082±0.018	0.102±0.020	0.110*±0.038	0.101±0.020
			Group 1 Air Control	Group 2 0.05 mg/m^3air	Group 3 0.25 mg/m^3air	Group 4 5.0 mg/m^3air
	After 52 Weeks of Recovery
	MALES	n	10	10	10	10
	BODY W. (g)	Mean± SD	594.7±78.6	668.9±78.5	660.0±97.7	694.2*±75.5
	BRAIN absolute (g)	Mean± SD	2.20±0.13	2.31 ±0.15	2.22 ±0.10	2.28±0.09
	LUNGS absolute (g)	Mean± SD	1.98±0.17	2.02±0.23	2.08±0.22	2.59**±0.20
	LUNGS % body weight	Mean± SD	0.34±0.03	0.30±0.03	0.32±0.04	0.38*±0.05
	LUNGS % brain weight	Mean± SD	90.47±9.91	87.32±8.86	93.74±8.56	113.77**±8.38
	LIVER absolute (g)	Mean± SD	17.01±1.78	19.25±2.76	18.48±2.98	21.05**±2.68
	LIVER % body weight	Mean± SD	2.88±0.26	2.88±0.24	2.81±0.28	3.05±0.34
	LIVER % brain weight	Mean± SD	777.58±105.62	829.73±79.44	838.19±159.81	925.58*±112.30
	THYMUS absolute (g)	Mean± SD	0.377±0.161	0.243±0.093	0.371±0.154	0.214*±0.049
	THYMUS % body weight	Mean± SD	0.062±0.020	0.036**±0.013	0.056±0.020	0.031**±0.007
	THYMUS % brain weight	Mean± SD	17.498±8.543	10.600±4.474	16.952±7.669	9.415*±2.213
	KIDNEYS absolute (g)	Mean± SD	2.66±0.20	2.98±0.39	2.76±0.30	3.03*±0.39
	KIDNEYS % body weight	Mean± SD	0.45±0.04	0.45±0.03	0.42±0.04	0.44±0.05
	KIDNEYS % brain weight	Mean± SD	121.51±12.52	128.32±10.30	124.95±14.07	132.91±14.82
	FEMALES	n	10	10	10	10
	BODY W. (g)	Mean± SD	343.2±42.6	383.7±48.8	376.8±71.2	410.5±74.7
	BRAIN absolute (g)	Mean± SD	2.06±0.11	2.11 ±0.13	2.04 ±0.07	2.14±0.13
	LUNGS absolute (g)	Mean± SD	1.43±0.08	1.54±0.11	1.51±0.12	2.24**±0.31
	LUNGS % body weight	Mean± SD	0.42±0.05	0.41±0.06	0.41±0.07	0.56**±0.12
	LUNGS % brain weight	Mean± SD	69.64±4.53	72.99±7.90	74.23±6.32	106.90**±10.39

 */**    DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**)

Text Table 4    Incidence and Severity of Test Item Related Microscopic Findings in the Lungs at the End of the 4- and 13-Week Exposure Periods and the 13- and 52 Week Recovery Periods ( / / / )

 

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/m^3Air)	0	0.05	0.25	5.0	0	0.05	0.25	5.0
n	0/10/10/10	0/10/10/10	0/10/10/10	5/10/10/10	0/10/10/9	0/10/10/10	0/10/10/10	5/10/10/10
Black particle deposition
trace	-/-/-/-	-/-/10/10	-/-/-/-	-/-/-/-	-/-/-/-	-/-/10/10	-/-/-/-	-/-/-/-
minimal	-/-/-/-	-/9/-/-	-/-/10/10	-/-/-/-	-/-/-/-	-/10/-/-	-/2/10/10	-/-/-/-
slight	-/-/-/-	-/-/-/-	-/10/-/-	1/-/-/-	-/-/-/-	-/-/-/-	-/8/-/-	3/-/-/-
moderate	-/-/-/-	-/-/-/-	-/-/-/-	4/-/2/-	-/-/-/-	-/-/-/-	-/-/-/-	2/4/3/-
marked	-/-/-/-	-/-/-/-	-/-/-/-	-/10/8/10	-/-/-/-	-/-/-/-	-/-/-/-	-/6/7/10
Mean Severity*	-/-/-/-	-/0.9/ 0.5/0.5	-/2.0/ 1.0/1.0	2.8/4.0/ 3.8/4.0	-/-/ -/-	-/1.0/ 0.5/0.5	-/1.8/ 1.0/1.0	2.4/3.6/ 3.7/4.0
Alveolar macrophages
minimal	-/-/-/-	-/-/-/-	-/8/10/10	-/-/1/6	-/-/-/-	-/-/-/-	-/9/10/9	-/-/-/3
slight	-/-/-/-	-/-/-/-	-/-/-/-	2/-/3/4	-/-/-/-	-/-/-/-	-/-/-/-	3/1/8/1
moderate	-/-/-/-	-/-/-/-	-/-/-/-	3/10/6/-	-/-/-/-	-/-/-/-	-/-/-/-	2/9//2/6
Mean Severity	na/-/-/-	na/-/-/-	na/1.0/ 1.0/1.0	2.6/3.0/ 2.5/1.4	na/-/-/-	na/-/-/-	na/0.9/ 1.0/0.9	2.4/2.9/ 2.2/2.3
Alveolar eosinophilic material
minimal	-/-/-/-	-/-/-/-	-/-/-/-	-/6/8/2	-/-/-/-	-/-/-/-	-/-/-/-	-/4/4/1
slight	-/-/-/-	-/-/-/-	-/-/-/-	-/2/2/3	-/-/-/-	-/-/-/-	-/-/-/-	-/4/5/1
moderate	-/-/-/-	-/-/-/-	-/-/-/-	-/1/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/1/-/5
Mean Severity	na/-/-/-	- na/-/-/-	na/-/-/-	-/1.3/ 1.2/0.8	na/-/-/-	na/-/-/-	na/-/-/-	-/1.5/ 1.4/1.8
Alveolar granulocyte infiltration
minimal	-/-/-/-	-/-/-/-	-/-/-/-	-/10/8/3	-/-/-/-	-/-/-/-	-/-/-/-	2/10/7/7
Mean Severity	na/-/-/-	na/-/-/-	na/-/-/-	-/1.0/ 0.8/0.3	na/-/-/-	na/-/-/-	na/-/-/-	0.4/1.0/ 0.7/0.7
Interstitial inflammation
minimal	-/-/-/-	-/-/-/-	-/-/-/-	5/-/1/9	-/-/-/-	-/-/-/-	-/-/-/-	5/4/5/9
slight	-/-/-/-	-/-/-/-	-/-/-/-	-/10/9/1	-/-/-/-	-/-/-/-	-/-/-/-	-/6/5/1
Mean Severity	na/-/-/-	na/-/-/-	na/-/-/-	1.0/2.0/ 1.9/1.1	na/-/-/-	na/-/-/-	na/-/-/-	1.0/1.6/ 1.5/1.1
Bronchiolar cell hypertrophy/ hyperplasia
minimal	-/-/-/-	-/-/-/-	-/-/-/-	5/10/-/1	-/-/-/-	-/-/-/-	-/-/-/-	5/10/-/4
slight	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/2
Mean Severity	na/-/-/-	na/-/-/-	na/-/-/-	1.0/1.0 /-/0.1	na/-/-/-	na/-/-/-	na/-/-/-	1.0/1.0/ -/0.8
Fibrosis, alveolar septa, focal/multifoca/focally extensive
minimal	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/3	-/-/-/-	-/-/-/-	-/-/-/-	-/-/2/5
slight	-/-/-/-	-/-/-/-	-/-/-/-	-/-/2/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/2
Mean Severity	na/-/-/-	na/-/-/-	na/-/-/-	-/-/0.4/0.3	na/-/-/-	na/-/-/-	na/-/-/-	-/-/0.2/0.9
Granulomatous fibrosing inflammation, focus(i)
minimal	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/1
slight	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/1
Mean Severity	na/-/-/-	na/-/-/-	na/-/-/-	-/-/-/-	na/-/-/-	na/-/-/-	na/-/-/-	-/-/-0.3
Increased lymphocytes, BALT
minimal	-/-/-/-	-/-/-/-	-/-/-/-	-/2/-/-	-/-/-/-	-/-/-/-	-/-/-/-	1/2/-/-
slight	-/-/-/-	-/-/-/-	-/-/-/-	-/1/-/-	-/-/-/-	-/-/-/-	-/-/-/-	-/-/-/-
Mean Severity	na/-/-/-	na/-/-/-	na/-/-/-	-/0.4/-/-	na/-/-/-	na/-/-/-	na/-/-/-	0.2/0.2/-/-

-       No animal affected, na: not applicable

*      Mean severity is ¿ number of animals x severity / number of examined organs in the group

 

Text Table 5    Incidence and Severity of Test Item Related Microscopic Findings in the Tracheobronchial Lymph Nodes at the End of the 13-Week Exposure and 13- and 52-Week Recovery Periods ( / / /)

 

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/m^3Air)	0	0.05	0.25	5.0	0	0.05	0.25	5.0
n	10/10/10	10/8/10	9/9/10	10/10/10	10/0/9	9/8/10	10/10/10	10/10/10
Black particle deposition
minimal	-/-/-	-/-/-	8/-/-	-/-/-	-/-/-	-/-/-	5/3/1	1/-/-
slight	-/-/-	-/-/-	-/5/5	6/3/1	-/-/-	-/-/-	-/-/2	7/1/1
moderate	-/-/-	-/-/-	-/2/-	4/7/3	-/-/-	-/-/	-/-/-	1/9/6
marked	-/-/-	-/-/-	-/-/-	4/7/4	-/-/-	-/-/-	-/-/-	1/9/3
Mean Severity*	-/-/-	-/-/-	0.9/1.0/ 1.0	2.4/2.7/ 2.7	-/-/-	-/-/-	0.5/0.3/ 0.5	1.8/2.9/ 3.2
Increased lymphocytes, cortex/paracortex
minimal	-/-/-	-/-/-	3/-/-	4/7/-	-/-/-	-/-/-	1/-/1	3/6/-
slight	-/-/-	-/-/-	-/-/-	3/2/-	-/-/-	-/-/-	-/-/-	7/-/-
moderate	-/-/-	-/-/-	-/-/-	3/-/-	-/-/-	-/-/-	-/-/-	-/-/-
Mean Severity	-/-/-	-/-/-	0.3/-/-	1.9/1.1/-	-/-/-	-/-/-	0.1/-/-	1.7/0.6/-
Endothelial vacuolation, high endothelial venule
minimal	-/-/-	-/-/-	1/-/-	1/2/-	-/-/-	-/-/-	-/-/-	2/5/-
slight	-/-/-	-/-/-	1/-/-	7/1/-	-/-/-	-/-/-	-/-/-	5/-/-
Mean Severity	-/-/-	-/-/-	0.3/-/-	1.5/0.4/-	-/-/-	-/-/-	-/-/-	1.2/0.5/-

-       No animal affected

*      Mean severity is ¿ number of animals x severity / number of examined organs in the group

 

Text Table 6    Incidence and Severity of Test Item Related Microscopic Findings in the Nasal Cavity at the End of the 13-Week Exposure and 13- and 52-Week Recovery Periods ( / / /)

 

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/m^3Air)	0	0.05	0.25	5.0	0	0.05	0.25	5.0
n	10/10/10	10/0/0	10/10/0	10/10/10	10/10/10	10/0/0	10/10/0	10/10/10
Eosinophilic globules, epithelium
minimal	4/2/4	-/na/na	2/3/na	2/6/1	-/3/1	-/na/na	3/2/na	1/4/4
slight	-/1/2	-/na/na	-/-/na	3/2/4	-/-/1	-/na/na	-/-/na	6/2/2
moderate	-/-/-	-/na/na	-/-/na	5/2/3	-/-/2	-/na/na	-/-/na	2/3/1
marked	-/-/-	-/na/na	-/-/na	-/-/2	-/-/-	-/na/na	-/-/na	-/-/1
Mean Severity*	0.4/0.4/ 0.8	-/na/na	0.2/0.3/ na	2.3/1.6/ 2.2	-/0.3/0.9	-/na/na	0.3/0.2/ na	1.9/1.7/ 1.5

-       No animal affected, na: not applicable

*      Mean severity is ¿ number of animals x severity / number of examined organs in the group

Text Table 7    Incidence and Severity of Noteworthy Findings in the Right Testis and Right Epididymis at the End of the 13-Week Exposure Period

 

Group	1	2	3	4
Dose (mg/m^3Air)	0	0.05	0.25	5.0
Right Testis
n	10	0	1	9
Seminiferous tubule atrophy/degeneration
minimal	4		-	1
slight	2		-	1
moderate	-		1	1
severe	-		-	1
Mean Severity*	0.8		na	1.2
Right Epididymis
n	10	0	1	9
Oligospermia
marked	-		1	2
Mean severity	0.0		na	0.9
Abnormal content
minimal	1		-	1
Mean Severity	0.1		na	0.1

N:     Number examined; -  : no animal affected ; na: not applicable

*:      Mean severity is ¿ number of animals x severity / number of examined organs in the group

 

Text Table 8    BALF, Cell Count

	Cell Count	Viability	Macro-phage	Eosino-phils	Lympho-cytes	Neutro-phils	Epithelial cells
	10^6	%	%	%	%	%
MALE
After 4 Weeks of Exposure
Group 1	1.60	83.60	95.9	0.0	2.7	1.1	0.3
Group 2	1.72	85.20	92.7	0.0	6.5	0.4	0.4
Group 3	2.04	82.00	89.2	0.0	5.3	4.2	1.2
Group 4	4.97**	90.63	36.4**	0.0	17.8**	45.3**	0.5
After 13 Weeks of Exposure
Group 1	1.74	84.40	93.9	0.0	4.4	1.5	0.1
Group 2	1.62	84.65	91.8	0.0	5.1	3.1	0.0
Group 3	2.24	89.00	64.8**	0.0	9.7*	25.2**	0.3
Group 4	2.01	88.00	31.5**	0.0	12.8**	55.6**	0.1
After 13 Weeks of Recovery
Group 1	1.80	90.10	96.5	0.0	2.1	1.2	0.2
Group 2	2.21	91.35	96.9	0.0	1.9	0.9	0.3
Group 3	2.85*	91.35	91.2	0.1*	3.8	4.7	0.2
Group 4	6.23**	89.86	44.6**	0.0	20.4**	39.8**	0.3
After 52 Weeks of Recovery
Group 1	1.50	87.35	97.1	0.0	2.6	0.2	0.1
Group 2	1.48	81.10*	94.8	0.0	3.6	0.6	0.8*
Group 3	1.40	87.45	95.4	0.0	2.4	1.7	0.5
Group 4	2.18	77.00**	77.8**	0.0	8.2**	13.6**	0.0
FEMALE
After 4 Weeks of Exposure
Group 1	3.19	89.40	96.7	0.0	2.7	0.5	0.1
Group 2	3.92	91.00	96.5	0.0	3.0	0.5	0.0
Group 3	3.70	87.30	94.9	0.0	4.0	0.9	0.1
Group 4	4.00	87.50	37.9**	0.0	15.3**	46.8**	0.0
After 13 Weeks of Exposure
Group 1	1.67	91.80	96.9	0.1	2.5	0.4	0.1
Group 2	1.27	92.55	94.5	0.0	4.4	1.0	0.1
Group 3	2.16	92.25	80.9**	0.0	4.1	15.0**	0.0
Group 4	2.98**	95.50	34.7**	0.0	11.5**	53.8**	0.0
After 13 Weeks of Recovery
Group 1	1.67	86.39	96.1	0.0	3.1	0.5	0.2
Group 2	1.66	85.40	95.8	0.0	3.4	0.8	0.0
Group 3	1.86	87.55	93.9	0.0	3.3	2.8	0.0
Group 4	4.02**	88.80	46.7**	0.0	13.0**	40.1**	0.0
After 52 Weeks of Recovery
Group 1	1.10	84.50	94.7	0.0	2.4	1.0	1.8
Group 2	1.34	82.95	97.9	0.0	1.4	0.6	0.2
Group 3	1.30	86.80	93.2	0.0	3.2	2.6	1.0
Group 4	2.06**	84.30	63.1**	0.0	15.8**	20.7**	0.2

*/**Significant at 5% (*), 1% (**)

Text Table 9    BALF, Biochemistry

	Phospholipids	Lactate dehydrogenase	Alkaline phosphatase	Gamma Glutamyl Transferase	Protein
	mmol/L	U/L	U/L	U/L	mg/L
MALE
After 4 Weeks of Exposure
Group 1	0.14	129.3	52.7	6.6	70.9
Group 2	0.18	144.2	45.7	6.3	68.8
Group 3	0.19	154.3	61.3	7.7	84.4
Group 4	0.88**	514.6**	124.8*	14.5	241.8
After 13 Weeks of Exposure
Group 1	0.16	196.8	37.6	5.9	92.7
Group 2	0.21	168.6	38.8	6.7	84.2
Group 3	0.36*	275.4	64.7	12.5**	115.4
Group 4	1.05**	1306.2**	195.4**	19.5**	395.0**
After 13 Weeks of Recovery
Group 1	0.26	225.7	76.4	5.6	115.4
Group 2	0.27	174.8	78.2	6.3	79.8
Group 3	0.42	237.2	108.5	9.5	102.3
Group 4	1.18**	1353.2**	330.3**	16.1**	347.4**
After 52 Weeks of Recovery
Group 1	0.21	104.9	43.5	4.8	51.2
Group 2	0.25	168.2	59.7	4.9	83.8
Group 3	0.25	114.3	49.0	5.5	63.9
Group 4	0.43**	619.5**	171.5**	12.4**	192.1**
FEMALE
After 4 Weeks of Exposure
Group 1	0.17	119.4	28.0	4.7	66.3
Group 2	0.17	95.1	21.2	4.3	66.1
Group 3	0.24	172.4	47.4	10.2	102.8
Group 4	0.68**	483.6**	94.1**	15.7*	235.7*
After 13 Weeks of Exposure
Group 1	0.17	163.6	34.9	5.1	78.8
Group 2	0.18	163.0	28.8	6.8	91.2
Group 3	0.30	312.7	63.4	12.5**	146.4
Group 4	0.82**	1020.2**	144.1**	15.9**	292.6**
After 13 Weeks of Recovery
Group 1	0.23	147.9	55.2	5.0	103.5
Group 2	0.22	159.0	45.3	4.8	90.8
Group 3	0.31	194.5	63.0	7.8	103.5
Group 4	0.76**	566.2**	117.8**	15.8**	300.2**
After 52 Weeks of Recovery
Group 1	0.15	92.7	30.4	3.8	57.5
Group 2	0.16	108.5	34.1	3.6	52.5
Group 3	0.20	111.2	39.0	6.1**	60.3
Group 4	0.50**	454.5**	98.4**	12.9**	186.6**

 

*/**Significant at 5% (*), 1% (**)

 

Text Table 10    cytokine Measurement in BALF

Mean ± SD (pg/mL)	IL-1ß	IL-5	TNF-a	IL-1 a
MALE

After 4 Weeks of Exposure
Group 1	11.16 ± 5.36	0.92 ± 1.35			1.37 ± 0.47	2.53 ± 5.66
Group 2	9.65 ± 2.90	1.51 ± 1.78			1.40 ± 0.66	< LoQ
Group 3	9.78 ± 4.04	< LoQ			2.34 ± 1.13	3.22 ± 3.48
Group 4	16.05 ± 8.20	0.84 ± 1.69			10.08± 3.66*	11.11 ± 22.22
After 13 Weeks of Exposure
Group 1	11.52 ± 6.43	< LoQ			1.34 ± 0.60	3.38 ± 6.40
Group 2	12.83 ± 9.11	< LoQ			1.47 ± 0.84	11.31 ± 11.70
Group 3	13.43 ± 5.96	< LoQ			4.56± 1.55**	5.99 ± 6.82
Group 4	10.60 ± 2.58	< LoQ			8.17± 2.66**	< LoQ
After 13 Weeks of Recovery
Group 1	15.68 ± 5.65	2.30 ± 4.87			1.83 ± 0.75	6.46 ± 4.20
Group 2	18.40 ± 3.69	7.12 ± 4.43*			2.21 ± 2.25	10.20 ± 8.39
Group 3	21.38 ± 6.63	12.38 ± 9.33**			4.46± 1.54**	9.82 ± 8.16
Group 4	24.64 ± 7.28**	4.63 ± 6.38			9.03± 2.28**	21.74± 24.45
After 52 Weeks of Recovery
Group 1	< LoQ	< LoQ			< LoQ	< LoQ
Group 2	< LoQ	< LoQ			< LoQ	< LoQ
Group 3	< LoQ	< LoQ			< LoQ	< LoQ
Group 4	< LoQ	< LoQ			< LoQ	38.09± 41.59*
FEMALES
After 4 Weeks of Exposure
Group 1	11.01 ± 3.59		0.38 ± 0.53	1.79 ± 0.69			< LoQ
Group 2	10.40 ± 4.64		< LoQ	2.44 ± 1.37			1.42 ± 3.18
Group 3	8.48 ± 1.97		< LoQ	2.60 ± 0.76			< LoQ
Group 4	20.08± 6.71		1.03 ± 2.3	11.38± 2.11**			< LoQ
After 13 Weeks of Exposure
Group 1	7.74 ± 2.90		1.34 ± 1.78	0.77 ± 0.20			7.15 ± 9.35
Group 2	6.31 ± 3.24		0.78 ± 1.27	0.80 ± 0.37			6.44 ± 10.70
Group 3	13.48 ± 5.58**		0.06 ± 0.18	6.53± 7.33**			11.94 ± 11.61
Group 4	13.81± 7.47*		0.57 ± 1.81	7.68± 1.35**			12.84 ± 16.71
After 13 Weeks of Recovery
Group 1	16.83 ± 4.57		13.41 ± 11.01	2.12 ± 0.85			9.94 ± 6.86
Group 2	15.63 ± 4.22		8.00 ± 3.54	1.89 ± 0.66			9.52 ± 10.59
Group 3	16.42 ± 3.66		4.98 ± 5.00*	4.54± 1.33**			6.75 ± 8.24
Group 4	24.57 ± 5.42**		1.46 ± 3.60**	11.16± 4.91**			4.49 ± 6.54
After 52 Weeks of Recovery
Group 1	< LoQ		< LoQ	< LoQ			< LoQ
Group 2	< LoQ		< LoQ	< LoQ			< LoQ
Group 3	< LoQ		< LoQ	< LoQ			< LoQ
Group 4	< LoQ		< LoQ	< LoQ			46.77± 25.76*

 

*/** Significant at 5% (*), 1% (**)

# Mean values higher than mean+2*sd (standard deviation) of the corresponding control group are in bold type (biologically significant)

LoQ Limit of Quantification

$ For the purpose of the mean and sd calculations, IL-1a level below the LoQ (13.7 pg/mL) were excluded from the calculations

Text Table 11    Sperm Count and Motility

 

	SPERM COUNT		MOTILITY
	TESTIS	CAUDA EPIDIDYMIS	PROGRESSIVE	STATIONARY	NON-MOTILE
	millions/g	millions/g	%	%	%
Historical Control Data (n = 6)	109.4 - 135.2	581.0 – 863.2	72.0 - 87.2	2.1 - 7.7	10.7 - 20.6
After 13 Weeks of Exposure
Group 1	111.8#	604.8#	71.5#	20.1#	8.3#
Group 2	127.4*	701.5	67.3	22.6	10.2
Group 3	136.6#**	835.5#**	62.8#	24.8#	12.4#*
Group 4	117.6#	597.9#	65.1#	24.2#	10.9#
After 13 Weeks of Recovery
Group 1	135.4#	775.8#	65.8#	27.1#	7.1#
Group 2			64.8	28.9	6.3
Group 3			61.0	31.4	7.7
Group 4	142.4	735.8	56.5 **	34.7**	8.8
After 52 Weeks of Recovery
Group 1	140.6°	594.4°	70.6	17.1	12.4
Group 2			66.6	16.0	17.4
Group 3			70.2	20.8	9.1
Group 4	138.4	595.0	74.9	17.5	7.6

*/**Significant at 5% (*), 1% (**)

§      Changes outside the historical control data are in bold type

#      Mean excluding animals no. 3 (control), 76 (group 3), 109 and 110 (group 4) of allocation A animals, as well as no. 18 (control) of allocation B animals presenting a testicular atrophy and a low sperm count.

°       Mean excluding animal no. 21 (control) of allocation C animals due to technical error.

 

Text Table 12    Sperm Morphology

 

	MORPHOLOGY
	A	B	C	D	E
	%	%	%	%	%
Historical Control Data	92.0 - 97.3	0.3 - 4.3	0.5 - 2.3	0.1 - 1.2	0.0 - 2.1
After 13 Weeks of Exposure
Group 1	91.6#	0.5#	4.7#	0.9#	2.3#
Group 2	92.4	1.3	3.7	0.2	2.5
Group 3	92.1#	0.7#	3.9#	0.7#	2.6#
Group 4	86.6#	0.9#	8.9#	1.2#	2.5#
After 13 Weeks of Recovery
Group 1	94.9#	1.0#	2.1#	0.5#	1.4#
Group 4	94.7	0.9	2.5	0.7	1.2
After 52 Weeks of Recovery
Group 1	96.1	0.5	1.4	0.5	1.4
Group 4	95.4	0.7	2.3	0.3	1.3

*/**Significant at 5% (*), 1% (**)

#      Mean excluding animals no. 3 (control), 76 (group 3), 109 and 110 (group 4) of allocation A animals, as well as no. 18 (control) of allocation B animals presenting a testicular atrophy and a low sperm count.

§     Changes outside the historical control data are in bold type

A     , complete sperm

B     head, abnormal tail

C     head only, tail detached

D     Abnormal head only, tail detached

E      Abnormal head, normal tail

 

Conclusions:

A 13-week rat inhalation toxicity study was performed with MWCNT Graphistrength™ C100. The milling procedure and the dust disperser used as solid aerosol generator produced an aerosol which retained the physico-chemical integrity of the original product in the test atmospheres. Principal health findings were limited to the lungs. The infiltration of phagocytizing macrophages is thought to be a trigger and results from the host reaction towards foreign bodies [Inhal Toxicol 2007, 19(Suppl 1):189-198]. The inflammation may also deteriorate the alveolar barrier function which increased particle translocation to the draining lymph nodes of the lung [Toxicol Sci 2010, 113(1):226–242]. Bronchial and alveolar epithelia were influenced secondarily. During an inhalation study, at a certain time point, a lung burden is reached that exceeds the macrophage clearance capacity and results in overload effects [Inhal Toxicol 2000, 12:1-17]. It seems to be the case at 5.0 mg/m3 air, as Graphistrength™ C100 deposition persisted in the lung without apparent signs of decrease after 3 and 12-month treatment-free periods, whereas at lower concentrations signs of clearance and recovery were observed. Prolonged TNF-a release in BALF was observed at 0.25 and 5.0 mg/m3 which was associated only at 5.0 mg/m3 with an increased collagen staining like that reported by Pauluhn [Toxicol Sci 2010, 113(1):226–242] with MWCNT Baytubes. The lack of microscopic change in the pleura indicated a lung reaction to Graphistrength™ C100 exposure quite different than that of the asbestos-like MWCNT-7 and could be related to the absence of internalization of Graphistrength™ C100 by the alveolar or mesothelial cells as shown by Tabet et al. [J Toxicol Environ Health A 2009, 72(2):60-73] with human epithelial A549 and mesothelial MeT5A cell lines cultures. Overall, these effects are consistent with a normal physiological response to the overload of the lung with insoluble particles [Toxicol Sci 2010, 113(1):226–242]. Considering the limited and reversible effects on the BALF parameters, the lack of pathological changes in the lungs and the clearance of the Graphistrength™ C100 observed at 0.25 mg/m3 air (0.279 mg/m3 analytical), this concentration can be considered as a No-observed Adverse Effect Concentration (NOAEC) for the local pulmonary toxiciy. The NOAEC for systemic toxicity was considered to be 5.0 mg/m3 (4.84 mg/m3 analytical).

Executive summary:

A key 13-week inhalation toxicity study in the rat with recovery periods was performed on Graphistrength™ C100 under a consent order according to the US Toxic Substances Control Act (TSCA) 5(e).

In this subchronic inhalation toxicity study, multiwalled carbon nanotubes Graphistrength C100 were administered 5 days per week by nose-only, flow-past inhalation to groups of 35 Wistar rats of both sexes at aerosol concentrations of 0.05, 0.25 and 5.0 mg/m³air (groups 2, 3 and 4, respectively) for a period of 13 weeks. A control group was treated similarly with air, only (group 1). An interim sacrifice was performed after 4 weeks of exposure (5 rats/sex/group, allocation D). Ten rats per sex and groups were sacrificed 24 hours after the last exposure (allocation A). Two sets of recovery animals were added to each group to investigate any delayed toxicity or reversibility. The first set of animals was necropsied after 13 weeks of recovery (10/sex/group, allocation B), the second set after 52 weeks of recovery (10/sex/group, allocation C).

Clinical signs, food consumption and body weights were recorded periodically for all animals during the acclimatization, exposure and recovery periods. Functional observation battery and ophthalmoscopic investigations were performed on allocation C rats at the end of the exposure period. Clinical laboratory investigations were performed during week 13 of exposure and at the end of the 13 and 52-week recovery periods on allocations A, B and C animals, respectively. Blood pressure of allocation C rats was measured during acclimatization, in week 13 of exposure (before and after exposure) and in week 52 of recovery. Vaginal smears were taken daily from all allocation B and C animals for 14 days during treatment weeks 11 and 12 and the estrus cycle was evaluated.

At the end of the corresponding period the animals were killed, necropsied and examined post mortem. Broncho-alveolar lavage was performed on all animals of allocations A to F. Broncho-alveolar lavage fluids from allocation A to D were investigated for cell count, viability, enzymatic activity and cytokines (lavage fluid from allocation F was discarded). Seminology and spermatid count was performed on males after 13 weeks of exposure and after 13 and 52 weeks of recovery. At the end of the 13-week treatment period, histological examination was performed on all tissues and organs from allocation A animals of groups 1 and 4 and on the respiratory tract organs from animals of groups 2 and 3. In addition, a histological examination was performed on the lungs of the animals of group 4 after 4 weeks of exposure. At the end of the 13- week recovery period, histological examination was performed on the lungs and tracheobronchial lymph nodes from all allocation B animals, the larynx and nasal cavity from animals of groups 1, 3 and 4 and the tracheal bifurcation from animals of groups 1 and 4. At the end of the 52-week recovery period, histological examination was performed on the lungs, pleura, heart and aorta from all allocation C animals, the tracheobronchial lymph nodes from animals of groups 1, 3 and 4 and the nasal cavity and tracheal bifurcation from animals groups 1 and 4. All gross findings were evaluated. Several tissues and samples were stored in glutaraldehyde and/or frozen for possible further investigation.

The target and achieved chemical aerosol concentrations over 13 weeks of dosing are presented in the following table.

 

Group	Target Aerosol Concentration [mg/m^3]	Gravimetric Aerosol Concentration [mg/m^3± SD]	Deviation from Target [%]
2 3 4	0.05 0.25 5.0	0.059 ± 0.042 0.279 ± 0.058 4.842 ± 0.408	+18.8% +11.7% -3.2%

 

The gravimetric mean mass median aerodynamic diameter (MMAD) was 1.54 and 2.30 µm in groups 3 and 4 and the mean count median aerodynamic diameter (CMAD) was 196, 231 and 208 nm in groups 2 to 4, showing that the generated aerosols were within the respirable range for rats for all groups.

Temperature, relative humidity and oxygen were considered to be satisfactory for this type of study.

The physico-chemical analysis and the transmission electronic microscopic observations of samples taken at different steps of the aerosol generation process did not show any substantial changes when compared to the original test item.

 

All animals survived the scheduled exposure period.

Hair loss, scabs, erythema and localized swelling were recorded. These signs are commonly seen in animals of this age and strain and are, therefore, considered to be incidental. Exposure to the test item had no effect on body weight and body weight gain development. There were no findings during functional observational battery and no effects on grip strength, body temperature, landing foot splay and locomotor activity. There were no differences in blood pressure and no ophthalmoscopic findings that were considered to be related with the test item.

Exposure-related changes in clinical laboratory parameters were noted exclusively for rats of group 4 and consisted of a shift in the differential white blood cell count characterized by an increase in neutrophil counts and a decrease in lymphocyte counts. These changes were considered to be secondary to the lung inflammatory reaction and were noted at the end of the 13-week exposure period and at the end of the 13-week recovery period in both sexes. At the end of the 52-week recovery period the changes were reversed in males, but not in females.

Increased potassium values were recorded in males of group 4 and in all treated groups of females at the end of the exposure period, but not at the end of the 13-week and 52-week recovery periods. No effects on urinalysis parameters and estrus cycle were recorded. There were no effects on sperm count, sperm motility and morphology.

An increase in IL-1ß levels in the broncho-alveolar lavage fluid (BALF) was observed in females of group 4 after 13 weeks of exposure. Additionally, the TNF-a levels in both genders of group 4 after 4 weeks of exposure, and groups 3 and 4 after 13 weeks of exposure and after 13 weeks of recovery were increased.

Test item-related necropsy findings were discoloration of and foci in the lungs as well as discoloration of the bronchial lymph nodes of rats at the high concentration at all necropsies.

After 4 and 13 weeks of exposurelung weights were increased in both sexes of group 4. Lung weights were still increased in group 4 after 13 weeks and 52 weeks of recovery.

Microscopic findings were as follows:

·        Deposition of black particles in the lungs after 4 weeks of exposure in group 4, and after 13 weeks of exposure, and after 13 and 52 weeks of recovery in groups 2 to 4;

·        Alveolar macrophages after 4 weeks of exposure in group 4, and after 13 weeks of exposure and after 13 and 52 weeks of recovery in groups 3 and 4;

·        Alveolar granulocyte infiltration and interstitial inflammation after 4 and 13 weeks of exposure and after 13 and 52 weeks of recovery in group 4;

·        Bronchiolar cell hypertrophy/hyperplasia and increased lymphocytes in bronchus associated lymphoid tissue (BALT) after 4 and 13 weeks of exposure and after 52 weeks of recovery in group 4;

·        Eosinophilic material in lungs of group 4 after 13 weeks of exposure and after 13 and 52 weeks of recovery;

·        Deposition of black particles at the tracheal bifurcation in group 4 after 13 weeks of exposure and after 52 weeks of recovery;

·        Focal/multifocal alveolar septae fibrosis in group 4 after 13 and 52 weeks of recovery;

·        Focal/multifocal granulomatous fibrosing inflammation and interstitial inflammation in the lungs of group 4 after 52 weeks of recovery;

·        Deposition of black particles in the tracheobronchial lymph nodes in groups 3 and 4 after 13 weeks of exposure and after 13 and 52 weeks of recovery;

·        An increase of lymphocytes within the cortex/paracortex of the tracheobronchial lymph nodes and vacuolation of the endothelial cells lining the high endothelial venules after 13 weeks of exposure in groups 3 and 4 and after 13 weeks of recovery in group 4.

Seminiferous tubule atrophy/degeneration was observed in the right testis of control and treated rats with a relatively high incidence and a similar severity. These effects were most likely related to the stress associated with immobilization in the restrainer during the inhalation procedure (Leeet al., 1993). All other findings were those commonly seen as spontaneous changes in the rat and bore no relationship to the test item. Of note, there were no exposure-related findings on the pleura, heart and aorta at the end of the 13-week exposure and 52-week recovery periods and on the olfactory bulb at the end of the 13-week exposure. Using brightfield optical and hyperspectral microscopy, in most of the lung samples of the exposed animals, the black agglomerates were identified as Graphistrength C100 with high fidelity. There was no Graphistrength C100 translocation in any of the distal organ tissues like brain, kidney and liver.

In view of black particle deposition in the lungs of rats associated at the high concentration of 5.0 mg/m³air with an inflammatory reaction still present after the 52-week recovery period, the No-Observed-Adverse-Effect Concentration (NOAEC) was established at the mid analytical concentration of 0.279 mg/m³. The NOAEC for systemic toxicity was considered to be 5.0 mg/m3 (4.84 mg/m3 analytical).

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Reference 2

Reference Type:

publication

Title:

Lung inflammation and lack of genotoxicity in the comet and micronucleus assays of industrial multiwalled carbon nanotubes Graphistrength(©) C100 after a 90-day nose-only inhalation exposure of rats

Author:

Pothmann D, Simar S, Schuler D, Dony E, Gaering S, Le Net JL, Okazaki Y, Chabagno JM, Bessibes C, Beausoleil J, Nesslany F, Régnier JF

Year:

2015

Bibliographic source:

Part Fibre Toxicol. 2015 Jul 10;12:21. doi: 10.1186/s12989-015-0096-2.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

GRAPHISTRENGTH C100

IUPAC Name:

GRAPHISTRENGTH C100

Constituent 2

Reference substance name:

Tangled Multi-Walled Carbon Nanotubes

EC Number:

701-160-0

Cas Number:

7782-42-5

Molecular formula:

Hollow tubular carbon, 1-dimensional nano structures with hexagonal arrangement of carbon atoms

IUPAC Name:

Tangled Multi-Walled Carbon Nanotubes

Test material form:

solid: nanoform

Test animals

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

Clean air

Details on exposure:

See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6-hours daily, 5 days/week at approximately 24-hour intervals

Post exposure period:

Approximately 24 hours after the last treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

(groups 2, 3 and 4, respectively)

Doses / concentrations 2

Dose / conc.:

0.05 other: mg/m³ air (target)

Doses / concentrations 3

Dose / conc.:

0.25 other: mg/m³ air (target)

Doses / concentrations 4

Dose / conc.:

5 other: mg/m³ air (target)

No. of animals per sex per dose:

5

Control animals:

other: yes, sham-exposed (group 1)

Positive control(s):

- Cyclophosphamide (CPA)
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: oral
- Doses / concentrations: single administreation of 20 mg/kg bw 24 hours before sacrifice

Examinations

Tissues and cell types examined:

Bone marrow cells (polychromatic erythrocytes)

Details of tissue and slide preparation:

Preparation of the Bone Marrow Smears
The femora were removed, the epiphyses cut off and the marrow will be flushed out with fetal calf serum, using a syringe (3 mL per femur). The nucleated cells were separated from the erythrocytes using the method of Romagna. Briefly, the cell suspensions were passed through a column consisting of a-Cellulose (Sigma) and Cellulose (Sigmacell type 50) (1:1 mixture). The columns were washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was resuspended in a small drop of FCS and spread on slides. The smears were air-dried, fixed in methanol and transported to Harlan CCR in Germany.
The slides were stained with May-Grünwald/Giemsa. For this at first the slides were incubated 1 minute in a 5% May-Grünwald solution followed by 1 minute in a 1 : 1 mixture of May-Grünwald : phosphate buffer (pH 7.4). After 20 minutes in 14% Giemsa solution the slides were washed twice in phosphate buffer and 10 seconds in deionised water. Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). Two slides were made from each bone marrow sample. One slide was shipped to Harlan CCR, the other was kept at Harlan Laboratories Ltd, Itingen. The slides were coded using a computer generated coding list.

Analysis of Cells
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of micronucleated polychro-matic erythrocytes nor a statistically significant positive response at any of the test points is con-sidered non-mutagenic in this system.

Statistics:

Nonparametric Mann-Whitney test, if necessary.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Bone marrow

Toxicity:

yes

Remarks:

inflammatory reactions in the lungs. No effect on the bone marrow

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Genotoxicity
No increase of the frequency of micronucleated polychromatic erythrocytes (PCE) and signs of medullar toxicity were observed in the rats after 13 weeks of exposure to Graphistrength™ C100 (Table). A clear positive response was observed with the positive control.

Any other information on results incl. tables

Table: Results of the micronucleus assay in the bone marrow of male and female rats exposed nose-only to respirable aerosols of Graphistrength™ C100 for 13 weeks

Test group (n=5)	Concentration mg/m³ air	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Males
Air control	0	24	0.340	2 -17	1099
Graphistrength™ C100	0.05	24	0.440	5 -11	1073
	0.25	24	0.280	2 -10	1094
	5.00	24	0.210	3 -8	1081
Positive control^1	20	24	0.833	10 -26	806
Females
Vehicle	0	24	0.290	3 -9	1155
Graphistrength™ C100	0.05	24	0.210	1 -8	1223
	0.25	24	0.190	1 -5	1147
	5.00	24	0.220	2 -9	1091
Positive control^1	20	24	0.750	8 -28	813

¹cyclophosphamide, 20 mg/kg

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The genotoxic potential of Graphistrength C100 was investigated in thein vivomicronucleus assay performed on the bone marrow cells of 5 male and 5 female Rcc HanTM: WIST(SPF) rats per groups around 24 hours following a nose-only, flow-past 13-week inhalation exposure (6 hours/day, 5 days/week) to 0, 0.05, 0.25 and 5.0 mg/m³air. Six animals/sex were treated with the positive control item cyclophosphamide. Per animal, 6000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the respective Air Control thus indicating that GRAPHISTRENGTH C100 did not exert any cytotoxic effects in the bone marrow in neither male nor female animals. In comparison to the respective Air Control values there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any sex and dose level after administration of the test item.Furthermore, no dose response was observed.In conclusion, GRAPHISTRENGTH C100 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat.