Tangled Multi-Walled Carbon Nanotubes

Endpoint summary

Description of key information

The repeated dose toxicity of Graphistrength® C100 has been evaluated in a 90-day inhalation toxicity (OECD 413) study and a 28-day dietary toxicity (OECD 407) study. Additional information are also provided by an inhalation OECD 421 study and a 5-day range-finding inhalation toxicity study.

Bronchioloalveolar inflammation characteristic of an overload with insoluble particles was observed after inhalation exposures for 4-7 and 13 weeks to a target concentration of 5.0 mg/m3 of Graphistrength™ C100 (Robinson, 2018; Broich, 2016). The severity (minimal or slight) was similar whatever the exposure duration.

 

Bronchioloalveolar inflammation

Target concentrations (mg/m^3)	0.05	0.25	1.25	5.0
Analytical concentration (mg/m^3) Schuler, 2010 Robinson, 2018 Broich, 2016	0.066 - 0.059	0.26 0.285 0.279	1.30 1.41 -	- 5.60 4.84
5 days (Schuler, 2010)	none	none	minimal/slight (=NOAEC)	-
4 -7 weeks (Robinson, 2018)	-	none	none (= NOAEC)	minimal (=LOAEC)
4 weeks (Broich, 2016)	none	none (= NOAEC)	-	minimal (=LOAEC)
13 weeks (Broich, 2016)	none	none (= NOAEC)	-	minimal/slight (=LOAEC)

 - not tested

 

At low concentrations, the severity of the effects did not increase between a 5-day and a 13-week exposure at 0.05 and 0.25 mg/m3 and at 1.25 mg/m3 between a 5-day and a 4/7-week exposure. Therefore, considering the large gap (x20) between the mid (0.25 mg/m3) and top (5.0 mg/m3) concentrations of the 13 -week toxicity study, the NOAEC for the local effects was established at 1.41 mg/m3 (analytical) (1.25 mg/m3 target) based on the OEC 421 study. Local effects on the respiratory tract are related to the deposited dose per unit of surface area. Below a certain concentration the clearance capacity of the lungs is not overwhelmed and the lungs retain the ability to clear the deposited MWCNT. Therefore, at this concentration, the lung effects are not suspected to worsen with increasing exposure duration (from sub-acute to chronic). A No-Observed Adverse Effect Concentration (NOAEC) of 4.84 mg/m3 (analytical concentration) was established for systemic toxicity after 13 weeks of exposure.

By oral route, no treatment-related effects indicating systemic toxicity were observed in male or female Sprague-Dawley rats exposed up to 10000 ppm of Graphistrength®C100 in diet, corresponding to mean dose levels of about 1000 mg/kg body weight/day.

 

90-day inhalation study with 3 and 12 -month recovery periods (OECD 413)

A key 13-week inhalation toxicity study in the rat with recovery periods was performed on Graphistrength® C100 (Broich, 2016; Pothmann et al., 2015; Régnier at al., 2017; Johnson, 2017). The purpose of this inhalation study was to assess the cumulative toxicity of Graphistrength® C100, to assess the pulmonary response by validated broncho-alveolar lavage methods, to indicate potential target organs and to evaluate a possible translocation from the lungs to distant organs like brain, liver and kidneys. The reversibility or progression of any test item related effects or any delayed toxicity was assessed after 13- and 52 -week treatment free recovery periods. This study was designed to provide a rational basis for the assessment of the toxicological risk to man.

Graphistrength® C100, was administered to four groups of 35 male and 35 female Wistar rats by nose-only, flow-past inhalation for a period of 13 weeks (6 hours/day, 5 days/week) to target concentrations of 0, 0.05, 0.25 and 5.0 mg/m3 (groups 1, 2, 3 and 4, respectively). A respirable dust aerosol was generated using a SAG 410 Solid Aerosol Generator connected to a micronizing jet mill and a cyclone and an elutriator thereafter. Previously, Graphistrength® C100 was ground in a ball mill for 12 hours under Argon and sieved at a mesh size of 63 µm, in order to facilitate aerosol generation. The milling procedure and the dust disperser used produced an aerosol which retained the physico-chemical integrity of the original product in the test atmospheres. A control group was treated similarly with air, only. An interim sacrifice was performed after 4 weeks of exposure. 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, the second set was necropsied after 52 weeks of recovery.

Clinical signs, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observation battery and ophthalmoscopic investigations were recorded at the end of the treatment period. Vaginal smears were performed for 14 days during treatment weeks 11 and 12 to evaluate the estrous cycle. Clinical laboratory investigations were performed during week 13 of treatment and at the end of the 1stand 2ndrecovery periods. In addition blood pressure was measured during acclimatization and in week 13 of treatment. At the end of the corresponding periods the animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. Broncho-alveolar lavages were performed on all animals for investigation of cell count, viability, enzymatic activity and cytokines. Semiology and spermatid count was performed on males after 13-weeks of treatment and 13- and 52-week of recovery. CytoViva brightfield optical and 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/m3of Graphistrength C100 for 13 weeks and after the 52-week treatment-free period.

The achieved aerosol concentrations over 13 weeks of dosing were 0.059, 0.279 and 4.84 mg/m3for target concentrations of 0.05, 0.25 and 5.0 mg/m3, respectively. The mean mass median aerodynamic diameter (MMAD) was between 1.57 and 2.29 µm, showing that the generated aerosols were within the respirable range for rats. 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 Graphistrength® C100.

All animals survived the scheduled treatment period. There were no clinical signs in any group. 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 Graphistrength® C100 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. Excepted increased potassium levels in males of group 4 and in all treated groups of females at the end of the treatment period, no other treatment-related changes were observed on the blood chemistry parameters. No effects on urinalysis parameters were recorded.

There was no difference in estrus cycle between treated and control groups. There was no treatment-related adverse effect on the sperm parameters at the end of the 13-week treatment and both recovery periods.

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. Exposure-related changes in the cell count of BALF were characterized by increased neutrophils, increased lymphocytes and decreased macrophages and were noted in rats of group 4 at all-time points and occasionally in rats of group 3 at the end of the 13-week exposure. 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. Exposure-related changes in clinical laboratory parameters of BALF were noted in rats of group 4 at all-time points and in rats of group 3 at the end of the 13-week exposure. They consisted of increased concentrations of phospholipids and protein and increased activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT). 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.

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 exposure, lung 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 (Lee et 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 conclusion, 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 0.279 mg/m3 air, this concentration can be considered to be the No-observed Adverse Effect Concentration (NOAEC) for local pulmonary effects and the NOAEC for systemic toxicity was considered to be 4.84 mg/m3 in this study.

 

Inhalation OECD 421 study

In an OECD 421 study (Robinson, 2018), groups of 10 male and female Han Wistar rats were exposed nose-only by inhalation, 6 hours per day, 7 days per week, for 2 weeks pre-pairing up to Day 19 post-coitum (pc) (a minimum of 4 weeks for the males) and from Day 5 of lactation until Day 12 to a respirable aerosol of Graphistrength C100 at actual concentrations of 0, 0.285, 1.41 and 5.60 mg/m3. The animals were checked twice daily for mortality, morbidity and clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 3, 7, 10, 14and 20 post-coitum (p.c.) and 1-3, 4-6 and 7-12 of lactation. Estrous cyclicity was checked for 15 days before pairing. Final body weights and selected organs weights (lungs, ovaries and male reproductive organs) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on larynx, lungs, tracheobronchial lymph nodes in all groups and on nasal passages, ovaries (with oviducts), epididymides and/or testes, pituitary in the control and high-dose groups, and on all macroscopic lesions.

Test item (black) staining on the head was recorded as a sign associated with dosing for all test groups, the incidence of this observation was related to exposure concentration. Wet fur seen on occasion is considered to be associated with the method of restraint. Test item (black) staining was also noted during the detailed weekly physical examinations on the head and forelimbs. No body weight gain was observed for females exposed to 5.60 mg/m3 compared with control over the first 2 weeks of exposures. During the gestation phase body weight gains for treated females were similar to control, during the lactation phase lower body weight gains were observed for all treated female groups, as low as 0.66X control; however this finding lacked exposure relationship and is thus considered to be unrelated to the test item. There were no effects on body weight gain for males. Food consumption was slightly low for females exposed to 1.41 or 5.60 mg/m3 over the first 2 weeks of exposures compared with control (approx. 0.9X control). Food consumption during gestation was similar to control for all treated female groups but was slightly lower than control for females exposed to 1.41 or 5.60 mg/m3 during lactation (0.9X control). There were no effects on food consumption for males. All thyroid hormone sampling were completed, T4 results from Day 13 pups and adult males are pending. Group mean lung and bronchi weight (unadjusted and adjusted) were higher than control for both sexes exposed to 1.41 or 5.60 mg/m3 , up to 1.3X for adjusted weights, for males and females (exposure related). Abnormal color (grey) was seen in the lungs of all animals exposed to 5.60 mg/m3 and in the majority of animals exposed to 1.41 mg/m3, in both sexes. Abnormal color (grey) was seen in the tracheobronchial lymph nodes of all females exposed to 5.60 mg/m3 and the majority of females exposed to 1.41 mg/m3. Dark lymph nodes were noted in some males exposed to 1.41 or 5.60 mg/m3. Enlargement was seen in the majority of males exposed to 5.60 mg/m3 and several males exposed to 1.41 mg/m3. Squamous metaplasia of the respiratory epithelium occurred in larynx of most females exposed to 5.60 mg/m3. Increased general cellularity of the tracheobronchial lymph node was observed in three males exposed to 5.60 mg/m3. Increased alveolar macrophages, with pigment, and bronchioloalveolar inflammation occurred in lungs of all males and females exposed to 5.60 mg/m3. Increased alveolar macrophages, with pigment, was also seen in most males and one female exposed to 1.41 mg/m3. In addition, an increased incidence of increased cellularity of the bronchus-associated lymphoid tissue (BALT) was recorded in some males and one female exposed to 5.60 mg/m3, when compared to controls. No treatment-related changes were observed at histological examination of the reproductive organs.

Under the experimental conditions and results of this study, the no-observed-adverse-effect concentration (NOAEC) for systemic toxicity was considered to be 5.60 mg/m3 and the NOAEC for local pulmonary toxicity was considered to be 1.41 mg/m3.

 

5-day range-finding inhalation study

In a dose range finding supporting study, Graphistrength® C100, was administered to three four of 20 male and 10 female Wistar rats by nose-only, flow-past inhalation for a period of five days (6 hours/day) to target concentrations of 0, 0.05, 0.25 and 1.25 mg/m3 (Schüler, 2010). A respirable dust aerosol was generated using a SAG 410 Solid Aerosol Generator connected to a micronizing jet mill and a cyclone and an elutriator thereafter. Previously, Graphistrength® C100 was ground in a ball mill for 20 hours and sieved at a mesh size of 63 µm, in order to facilitate aerosol generation. Throughout the study all animals were observed for viability and clinical signs. In addition, body weight and food consumption were recorded. Subgroups of five males and five females were sacrificed 24 hours after at the last exposure and after a 4-week treatment-free period and subject to a macroscopic examination. Adrenals glands, heart, kidneys, liver, lungs and spleen were weighed and heart, kidneys, larynx, liver, lungs, mediastinal lymph nodes, nasal cavity, naso pharynx, spleen, trachea and all gross lesions were examined microscopically. Additionally, 5 males were also sacrificed at the same time schedule for cytological (cell count, viability and differential cell counts) and biochemical (LDH, ALP, GGT and proteins) examinations of the broncho-alveolar lavage (BAL) fluids. Exposure to gravimetrically determined aerosol concentrations of 0.066, 0.26 and 1.30 mg Graphistrength® C100 mg/m3air were achieved in groups 2 to 4, respectively. The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined Mass Median Aerodynamic Diameters (MMAD) of 1.93 - 2.56, 1.97 and 1.84 - 1.85 µm and Geometric Standard Deviations (GSD) of 1.30 - 4.33, 1.94 and 1.76 - 1.93, respectively. Mean percentages of particles lower than 3 µm were 68.4, 73.6 and 78.3%, respectively. The generated aerosols were considered to be acceptable for inhalation toxicity testing in rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study. All animals survived to the end of the scheduled treatment period and no clinical signs were observed. There were no test item-related effects on food consumption or body weight. A slight increase in neutrophil count was observed at 1.30 mg/m3 (2.8% vs. 0.3% in controls) after the 5-day treatment, which disappeared after the 4-week recovery period. GGT levels were statistically significantly increased at 1.30 mg/m3 (11.4 vs. 3.2 U/L) but were normal after the recovery and protein values were statistically significantly increased at 0.26 and 1.30 mg/m3 after exposure (0.95 and 0.83 vs. 0.04 g/L) and after the 4-week recovery period (0.64 and 0.75 vs. 0.21 g/L). There were no effects on organ weights or macroscopic findings which were considered to be related to treatment. Treatment-related microscopic findings were noted in the lungs of male and female rats, both after the 5-day exposure and the recovery period. Dose-dependent increased black inclusions were observed in the cytoplasm of infiltrated macrophages, indicating an adequate exposure of the lungs. Macrophage infiltration of the lung (grade 2) was observed at 1.30 mg/m3in 3 males and 3 females after the 5-day exposure and in 4 males and 4 females after the recovery period. Hypertrophy of the bronchial and bronchiolar cells was observed at 1.30 mg/m3in 4 males (3 grade 1 and 1 grade 2) and 2 females (1 grade 1 and 1 grade 2) after the 5-day exposure and in 2 males and 2 females (all grade 1) after the recovery period. No treatment-related microscopic findings were observed in the other organs examined. In conclusion, a 5-day exposure of rats to 1.30 mg/m3of a respirable aerosol of Graphistrength® C100 induced effects on the lungs, partially reversible after a 4-week treatment free period. On the basis of these findings, a No-Observed-Adverse-Effect-Concentration (NOAEC) of 1.30 mg/m3 air may be established for both systemic and local pulmonary toxicity.

 

A “disregarded” 5-day range finding study was carried out with Graphistrength® C100 dust aerosol, to determine the pulmonary toxicity (Ma-Hock et al., 2013). Eleven male Wistar rats per test group and time point were head-nose exposed to respirable dusts for 6 hours per day, on 5 consecutive days. The target concentrations were 0.1, 0.5 and 2.5 mg/m3. A concurrent control group was exposed to conditioned air. Animals were sacrificed on study day 7 and 28. On each sacrificing day 5 animals per group underwent lung lavage and six animals per group were designated for histopathological examination. The concentrations were produced with brush particle generators. The dust concentration was determined by gravimetrical measurements. The exposure of rats to Graphistrength® C100 caused concentration dependent pulmonary inflammation as indicated by biochemical parameters in BALF and the increased weights of lung and histopathology. The effect is not reversible. Under these test conditions, the NOAEL was 0.1 mg/m3. After completion of the study, samples of the test atmosphere collected in cyclonic separator and at exhaust of the inhalation chamber were analysed by Electron Spectroscopy for Chemical Analysis (ESCA) and Transmission Electronic Microscopy (TEM) in comparison with the original batch of the Graphistrength® C100 used in this study (Quet, 2008; Pothmann et al., 2015). There was very little oxygen function in surface of the original sample (close to the detection limit). By cons, there is a significant oxidation of the surface of samples collected from the elutriator (x5) and from the inhalation chamber (x10). Taking into account that the ESCA analysis gives an average composition of ten graphite planes, this means that if oxygen is grafted onto a single external graphitic plane, this corresponds to a content of 20 oxygen atom %. A high TEM magnification observation showed that some MWCNTs from the inhalable fraction were heavily modified by mechanical brushing with the formation of holes in the walls. Portions of MWCNTs resembled lace. Generally the MWCNT did not seem to have been cut. Therefore, the increased percentage of oxygen at the surface can be due to the mechanical damage. As mentioned in the discussion of the Ma-Hock’s article, structural defects are an important factor governing biological effects of MWCNTs. Therefore, it was demonstrated that the brush dust generator used in this study induced an oxidation of the MWCNTs and a significant physical destruction of the structure of the MWCNTs, this did not occur with the generator used by Schuler in a 5-day inhalation toxicity study. In consequence, the toxicological response to particles generated with the brush dust generator was far more severe than to particles generated with the solid aerosol generator/micronizing jet mill. In conclusion, the results reported in the article by Lan Ma-Hock et al. are not considered to be reliable to assess the toxicological properties of the Graphistrength® C100.

 

28-day dietary administration (OECD 407)

The toxicity of Graphinstrength C100 MWCNT was investigated in Spraque Dawley rats after 4 weeks when given orally, via diet (Monetini, 2019). The animals were assigned to four groups of 5 animals/sex and received the test item, mixed in the rodent diet, at fixed concentrations of 100, 1000 and 10000 ppm, corresponding to mean achieved dose levels over 4 weeks of 10, 95 and 951 mg/kg body weight/day for males and 11, 105 and 1073 mg/kg body weight/day for females, respectively. Routine in vivo analyses included daily clinical observations and food consumption. At weekly interval, body weight and assessment of detailed clinical observations (removal from the home cage and observation in open arena) were performed. In addition, towards the end of treatment specific functional tests (hindlimb landing foot splay, sensory reactivity to stimuli including grip strength, and motor activity) were performed for neurotoxicity assessment. Oestrous cycle was also evaluated on Day 29, before dispatch to necropsy. Bleeding for clinical pathology investigation including urinalysis was carried out. At term, the animals were subjected to a detailed macroscopic examination along with organ weights and tissue retention.

No mortality occurred in the study. The main clinical signs were limited to a general black discolouration of the body surface due to the black colour of the test item. Additionally, at observation of the cage tray, black staining was also recorded in the mid- and high dose groups. Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups. Comparable body weight values were recorded in the control and treated groups through the study. Food consumption did not show relevant changes through the study. No treatment-related effects were seen at hematology or clinical chemistry analyses. The presence of black staining in the urine sediment was detected and associated with the presence of large MWCNT aggregates due to an external contamination of the urine by the test item retained in the fur and/or excreted in feces.The dark discolouration of skin, muzzle and/or tail was likely due to the colour of the test item. No changes were recorded at histopathological examination of tissues that could be related to the test item. In conclusion, no treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated.

 

Intranasal, oropharyngeal injection

A synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidyl glycerol, cholesterol and bovine serum albumin (BSA), was elaborated as a vehicle to study the lung toxicity of Graphistrength™ 100 in mice (1.5, 6.25 and 25 µg/mouse) (Ronzani et al., 2012). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice upon repeated administrations (on days 0, 7 and 14) was analyzed by histology and TEM on day 21. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF) -ß1 and collagen, and by histology. Surfactant dispersed MWCNT distributed all throughout the mouse airways and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. After repeated MWCNT administrations, increases in macrophage number, KC and TGF-ß1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed. 

 

Pulmonary exposure to nanoparticles MWCNT may affect, in addition to pulmonary toxicity, the cardiovascular system such as procoagulant effects, vascular dysfunction and progression of atherosclerosis (Luyts et al., 2014). Bmal1 (brain and muscle ARNT-like protein-1) knockout (Bmal1(-/-)) mice which have a disturbed circadian rhythm and procoagulant phenotype were used to study the pulmonary and hemostatic toxicity of Graphistrength C100 after subacute pulmonary exposure. Bmal1(-/-) and wild-type (Bmal1(+/+)) mice were exposed via oropharyngeal aspiration, once a week, during 5 consecutive weeks, to a cumulative dose of 32 or 128µg MWCNTs. Graphistrength C100 caused a pronounced inflammatory response in the lung with increased cell counts in the broncho-alveolar lavage and increased secretion of interleukin-1ß and cytokine-induced neutrophil chemo-attractant (KC), oxidative stress (increased ratio of oxidized versus reduced glutathione and decreased total glutathione) as well as anemic and procoagulant effects as evidenced by a decreased prothrombin time with increased fibrinogen concentrations and coagulation factor (F) VII. The Bmal1(-/-) mouse is a sensitive animal model to study the procoagulant effects of MWCNTs, the correlation analysis suggests a causal association between the observed pulmonary and procoagulant effects. 

 

The vascular effects of Graphistrength C100 was evaluated in apolipoprotein E-/- mice and wild-type mice (Cao et al., 2014). The ApoE-/- mice had accelerated plaque progression in aorta after 5 intratracheal instillations of MWCNT (25.6µg/mouse weekly for 5 weeks). The exposure was associated with pulmonary inflammation, lipid peroxidation, and increased expression of inflammatory, oxidative stress, and vascular activation response genes. Despite up regulation of inflammatory genes in the liver, effects on systemic cytokines and lipid peroxidation were minimal. 

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 October 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 6 weeks
- Weight at study initiation: males 233-259 g, females: 167-197g
- Fasting period before study: none
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet (ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l.)
- Water: ad libitum
- Acclimation period: approximately 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C± 2
- Humidity (%): 55%±15
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The test item Graphistrength C100 will be formulated, using powdered diet, by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing. The formulations will be prepared separately for each group. Fresh diets will be prepared at weekly intervals, unless specified otherwise, at fixed concentrations of 100, 1000 and 10000 ppm. Concentrations will be calculated and expressed in terms of test item as supplied.
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis has been performed by ICP-OES (inductively coupled plasma optical emission spectrometry) of Al and Fe concentrations resulting from the residual amount of catalist. Samples have been mineralized beforehand by alkali fusion or micro-wave digestion. Some tests have also been conducted by XRF (X-ray fluorescence). However, the analysis couldn’t confirm the Graphistrength® C100 concentrations in the diet formulations containing nominally 100, 1000 and 10000 ppm of Graphistrength® C100. The main issue comes from the low Graphistrength® C100 concentrations to measure (and so the very low Al and Fe concentrations) in rat diet formulations that contains already non negligible Al and Fe amounts.
Due to the uncertainty of the measurements (with Alkali fusion + ICP-OES), concentrations of 100 and 1000 ppm of Graphistrength® C100 have no chance to be detected. The 10000 ppm Graphistrength® C100 concentration is the only one that could be quantified but the recovery (ca. 72%) was lower than the expected values.

Duration of treatment / exposure:

28 days

Frequency of treatment:

ad libitum

Dose / conc.:

100 mg/kg diet

Dose / conc.:

1 000 mg/kg diet

Dose / conc.:

10 000 mg/kg diet

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day

DETAILED CLINICAL OBSERVATIONS: Yes
Observations were performed at the same time interval each day. Observation of the cage was also performed. The data are not presented in this report but retained as study raw data. Once before commencement of treatment and once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
Once during Week 4 of treatment, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.

BODY WEIGHT: Yes
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of rat was recorded daily, starting from the allocation. Due allowance was made for any spilled food in each cage. The group mean daily intake per rat was calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of Week 4 of treatment
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Heinz bodies
– Differential leucocyte count
· Neutrophils
· Lymphocytes
· Eosinophils
· Basophils
· Monocytes
· Large unstained cells
– Platelets
– Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of Week 4 of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Bile acids
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: end of Week 4 of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
– Appearance
– Volume
– Specific gravity
– pH
– Protein
– Glucose
– Ketones
– Bilirubin
– Urobilinogen
– Blood
Part of the sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined, microscopically for:
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components (e.g. the presence of Multiwalled carbon nanotubes, MWCNT)

NEUROBEHAVIOURAL EXAMINATION: Yes
The motor activity (MA) of all animals was measured once during Week 4 of treatment by an automated activity recording. Measurements were performed using a computer generated random order.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).

ORGAN WEIGHT: Yes
See Table TISSUE PROCESSING

HISTOPATHOLOGY: Yes
See Table TISSUE PROCESSING

Other examinations:

CytoViva hyperspectral microscopy was employed to spectrally identify Graphistrength C100 multiwalled carbon nanotubes (MWCNT) in H&E stained kidney and urinary bladder tissues of Sprague-Dawley rats exposed to 10,000 ppm of Graphistrength C100 MWCNT via diet for 4 weeks. The samples were imaged with transmitted brightfield. Each tissue sample was surveyed at 10x magnification under brightfield illumination to identify any dark particles that appeared to be Graphistrength C100 MWCNT. As no dark deposits were found in any samples, 60x magnification hyperspectral and optical images were acquired at random areas in the tissue. No mapping was required because no dark deposits were found.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicological relevance were recorded. Animals of the mid- and high dose groups showed black appearance of the general body surface including tail (see attached pictures).
Additionally, at observation of the cage tray, black staining was also recorded in the mid- and high dose groups. One low dose animal (no. 20) showed moderate decreased activity, piloerection and hunched posture during the last days of study. Mild ulceration in the stomach was recorded at histological examination that might account for the presence of such clinical signs. No other relevant signs were recorded in the low dose group.
Weekly observation of animals at removal from the cage and in an open arena (open-field assessment) did not reveal changes attributable to the test item.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean values of the achieved dosage, calculated over the first two weeks, were 11, 104 and 1003 mg/kg/day for males and 11, 111 and 1152 mg/kg/day for females, respectively for the low, mid- and high dose level. During the last two weeks, the mean values of the achieved dosage were 9, 86 and 900 mg/kg/day for males and 10, 99 and 995 for females, for the low, mid- and high dose level respectively. The overall achieved dosages for the 4 weeks of study were 10, 95 and 951 mg/kg/day for males and 11, 105 and 1073 mg/kg/day for females.

Haematological findings:

no effects observed

Description (incidence and severity):

Some statistically significant changes were recorded in treated animals, such as: increase of erythrocytes, haemoglobin and haematocrit in males dosed at 100 ppm, increase of haemoglobin and haematocrit in those receiving 10000 ppm and decrease of mean corpuscular haemoglobin concentration in females treated at 100 ppm. Due to the minimal severity (3% to 8%) and/or the absence of dose-relation, the above findings were considered incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Alkaline phosphatase was reduced in some females dosed at 100 ppm and 10000 ppm. Mean group values were 25% and 21% below controls, respectively. In addition, male no. A2321030 (1000 ppm) showed high aspartate aminotransferase value (69% above mean control data). Due to the minimal incidence and/or the absence of dose-relation, the above findings were considered incidental.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination of fresh samples of urine sediment revealed the presence of few dark amorphous aggregates for three males and two females dosed at 10000 ppm. As additional analysis, the urine sediments were further investigated by the Sponsor (Godillot, 2017), these formations were identified by scanning electronic microscopy as MWCNT agglomerates of several tens of micrometers (Godillot, 2017).

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant changes were noted in treated animals at post mortem, when compared with control animals. The changes observed in treated groups of both sexes, such as brown staining of the head (muzzle) or dark colour of skin and/or tail are suggested to be likely due to the colour of the test item. The remaining sporadic findings observed in few treated animals could be considered incidental.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No changes or traces related to the treatment with Graphistrength C100 were noted in the organs/tissues. All observed lesions are known to occur spontaneously in Sprague Dawley SD rats of the same age and/or have a comparable incidence in the control group.
As additional analysis, kidneys and urinary bladder tissue samples were further investigated by the Sponsor using brightfield hyperspectral microscopy (Jonhson, 2017). No trace of Graphinstrength C100 MWCNT was observed which could account for the MWCNT agglomerates observed in the urine sediments of some rats exposed to the top concentration.

Other effects:

no effects observed

Description (incidence and severity):

Oestrous cycle
Evaluation of the oestrous cycle at the end of study (Day 29) did not indicate particular differences between groups.

Dose descriptor:

NOAEL

Effect level:

>= 10 000 mg/kg diet

Based on:

test mat.

Remarks:

ca. 1000 mg/kg bw/d

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

A 4 week toxicity study on Graphistrength C100 was conducted in Spraque Dawley rats. The test item was given orally, via diet, at constant concentrations of 100, 1000 and 10000 ppm, corresponding to mean achieved dose levels over 4 weeks of 10, 95 and 951mg/kg body weight/day for males and 11, 105 and 1073mg/kg body weight/day for females, respectively.
The in-life phase did not show changes of toxicological relevance. The most remarkable clinical signs observed in treated animals of the mid- and high dose groups was dark staining of the general body surface including tail resulting from the accumulation of MWCNT in the fur after the contamination of the litter by spillage of diet and/or excretion of MWCNT in the feces. In addition, data collected from detailed multiple functional observation battery tests and specific functional tests including motor activity, landing foot splay and sensory reactivity to stimuli, did not show evidence of neurotoxic effects of the test compound.
Body weight and food consumption were unaffected by treatment.
At clinical pathology investigations, no important changes were recorded in hematological, biochemistry or urinalysis parameters that could be considered as treatment-related. The presence of large dark aggregates was observed in the urine sediment of many high dose animals, which were identified as MWCNT by scanning electron microscopy (Godillot, 2017).
At necropsy examination, the macroscopic findings were limited to dark soft content of caecum and stomach or dark colour of skin and/or tail in treated animals. The remaining sporadic findings observed in few treated animals could be considered incidental.
The histological analysis did not reveal changes related to inflammatory reactions or cell injury in any organ/tissues. The remaining sporadic findings observed in few treated animals could be considered incidental.
A specific histological investigation was performed by CytoViva© Inc. (Johnson, 2017) using hyperspectral microscopy to spectrally identify any dark particles that could appear to be Graphistrength C100 in H&E stained kidney and urinary bladder tissues of rats exposed to 10000 ppm. No dark deposits were found in ant tissue samples. The results support the conclusion that the MWCNT observed in some urinary sediments was due to an external contamination (via the fur and/or feces) during the urine collection.
In conclusion, no signs of toxicity were observed in male or female animals at any of the dose levels investigated.

Executive summary:

The toxicity of Graphinstrength C100 MWCNT was investigated in Spraque Dawley rats after 4 weeks when given orally, via diet. The animals were assigned to four groups of 5 animals/sex and received the test item, mixed in the rodent diet, at fixed concentrations of 100, 1000 and 10000 ppm, corresponding to mean achieved dose levels over 4 weeks of 10, 95 and 951 mg/kg body weight/day for males and 11, 105 and 1073 mg/kg body weight/day for females, respectively. Routine in vivo analyses included daily clinical observations and food consumption. At weekly interval, body weight and assessment of detailed clinical observations (removal from the home cage and observation in open arena) were performed. In addition, towards the end of treatment specific functional tests (hindlimb landing foot splay, sensory reactivity to stimuli including grip strength, and motor activity) were performed for neurotoxicity assessment. Oestrous cycle was also evaluated on Day 29, before dispatch to necropsy. Bleeding for clinical pathology investigation including urinalysis was carried out. At term, the animals were subjected to a detailed macroscopic examination along with organ weights and tissue retention.

No mortality occurred in the study. The main clinical signs were limited to a general black discolouration of the body surface due to the black colour of the test item. Additionally, at observation of the cage tray, black staining was also recorded in the mid- and high dose groups. Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups. Comparable body weight values were recorded in the control and treated groups through the study. Food consumption did not show relevant changes through the study. No treatment-related effects were seen at hematology or clinical chemistry analyses. The presence of black staining in the urine sediment was detected and associated with the presence of large MWCNT aggregates due to an external contamination of the urine by the test item retained in the fur and/or excreted in feces. The dark discolouration of skin, muzzle and/or tail was likely due to the colour of the test item. No changes were recorded at histopathological examination of tissues that could be related to the test item. In conclusion, no treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Key study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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).

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHan™ WIST

Sex:

male/female

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.4 - 3 µm

Geometric standard deviation (GSD):

2.38

Dose / conc.:

0.25 other: mg/m3 air (target conc.)

Remarks:

0.285 mg/m3 (analytical)

Dose / conc.:

1.25 other: mg/m3 air (target conc.)

Remarks:

1.41 mg/m3 (analytical)

Dose / conc.:

5 other: mg/m3 air (target conc.)

Remarks:

5.60 mg/m3 air (analytical)

Control animals:

yes, sham-exposed

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

5.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEC

Remarks:

Local effect

Effect level:

1.41 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

5.6 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

5-day range finding study including a 4 week treatment free recovery period

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at Delivery): 9 weeks
- Body Weight Range (at Acclimatization):
Males: 256.0 to 289.5 g (±8%)
Females: 140.2 to 184.1 g (±15%)
- Fasting period before study: no
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (ad libitum): Pelleted standard Harlan Teklad 2914C
- Water (ad libitum): Community tap-water
- Acclimation period: Fourteen days

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined Mass Median Aerodynamic Diameters (MMAD) of 1.93 - 2.56, 1.97 and 1.84 - 1.85 µm and Geometric Standard Deviations (GSD) of 1.30 - 4.33, 1.94 and 1.76 - 1.93, respectively. Mean percentages of particles lower than 3 µm were 68.4, 73.6 and 78.3%, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: the animals are confined separately in restraint tubes
- System of generating particulates/aerosols:
Prior to use for aerosol generation, the test item was ground in a ball mill for 20 hours. The resulting fine particles were used in the present study. Investigations on the milling process were performed under Harlan Laboratories Study C32562: No surface oxidation (increase of O2 content) was noted after 24 hours of milling and transmission electron microscopy of aerosol filter sampling demonstrated that milling and aerosol generation did not damage the surface of GRAPHISTRENGTH C100.
A dust aerosol was generated from the 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. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The aerosol concentrations for groups 2 and 3 were achieved by serial dilution with compressed, filtered, dry air of the aerosol generated for group 4, using an air vacuum device.
- Temperature, humidity, pressure in air chamber:
Aerosol concentration, particle size distribution, relative humidity, temperature and oxygen concentration were measured on test aerosol samples taken at a representative exposure port.
- Air flow rate: flow of air at each tube was between 0.73 to 1.0 L/min
- Method of particle size determination:
The cumulative particle size distribution of the test aerosol was determined using an 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 twice for groups 2 and 4 and once for group 3. The airflow rate through the impactor was approximately 9 L/min. The difference to the lower flow of aerosol air at each tube was considered to be justified in order to increase the impact of the aerosol particles having a low density. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD)
were calculated on the basis of the gravimetric results from the impactor.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
Test aerosol samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The duration of sampling was sufficient to ensure reliable results. Gravimetric determination of the aerosol concentration was performed at twice daily for groups 2 and 3 and once daily for group 4. Sampling flow rate was 3 to 4 L/min per exposure port. During technical trials it was confirmed that the difference to the lower flow of aerosol air at each tube did not affect the results but increased the accuracy of the measurement.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours/day

Dose / conc.:

0.05 other: mg/m3 (target)

Remarks:

0.066 mg/m3 (analytical)

Dose / conc.:

0.25 other: mg/m3 (target)

Remarks:

0.26 mg/m3 (analytical)

Dose / conc.:

1.25 other: mg/m3 (target)

Remarks:

1.30 mg/m3 (analytical)

No. of animals per sex per dose:

20 males and 10 females

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Target concentrations were selected based on published evidence on carbon nanotube
- Rationale for selecting satellite groups:
Allocation and Aerosol Concentrations
mg/m3 air Group 1 Group 2 Group 3 Group 4
control* 0.050 0.25 1.25
0
Males A 1 - 5 21 - 25 41 – 45 61 - 65
B 6 - 10 26 - 30 46 - 50 66 - 70
C 11 – 15 31 – 35 51 – 55 71 – 75
D 16 - 20 36 - 40 56 - 60 76 - 80
Females A 81 – 85 91 – 95 101 – 105 111 – 115
B 86 - 90 96 - 100 106 - 110 116 - 120

* Control animals were treated with air only
A Main study animals
B 4-week recovery animals
C Animals (males) for BAL
D 4-week recovery animals (males) for BAL

- Post-exposure recovery period in satellite groups: 4 weeks

Observations and examinations performed and frequency:

VIABILITY / MORTALITY: Yes
Twice daily during treatment and once daily during acclimatization and recovery.

CAGE SIDE OBSERVATIONS: Yes
Cage-side clinical observations once daily during treatment and recovery. Twice weekly during acclimatization.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Recorded twice weekly (each individual animal) during acclimatization and at days 1 and 5 of the treatment. Weekly during recovery.

FOOD CONSUMPTION:
Recorded twice weekly (per cage) during acclimatization and treatment. Weekly during 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: Yes / No / No data

WATER CONSUMPTION: No:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.

ORGAN WEIGHT: Yes
Adrenal glands, Heart including auricles, Kidneys, Liver, Lungs, instilled via trachea with formalin at approximately 30 cm H2O pressure, Spleen

HISTOPATHOLOGY: Yes
All gross lesions, Heart including auricles, Kidneys, Larynx, Liver, Lungs, Lymph nodes - mediastinal, Nasal cavity, Nasopharyngeal duct and pharynx, Spleen, Trachea - adjacent to larynx and carina and bifurcation

Other examinations:

BRONCHO-ALVEOLAR LAVAGE FLUID (BALF)
5 male animals from each group were sacrificed approximately 24 h after the last (5-day) exposure and after 4 weeks of recovery (Allocation C and D).
Parameters: enzymatic activity of lactate dehydrogenase, alkaline phosphatase, and gamma-Glutamyltransferase, total protein, total cell count, cell viability and differential cell counting (macrophages, neutrophils, lymphocytes, eosinophils, epithelial cells, and other cells).

Statistics:

The following statistical methods were used to analyze the food consumption, body weight, macroscopic findings, organ weights and ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied 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 instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were noted during the course of the study.

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled treatment or recovery periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight which were considered to be related to exposure to the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food intake was similar across all groups during the study.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute kidney weight in males of groups 3 and 4 was statistically significantly reduced as well as the kidney to body weight ratio of group 3. No dose-dependency was noted.
There were no further effects on organ weights which were considered to be possibly related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross lesions that could be attributed to treatment with the test item.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lungs
In the main study animals, increased severity of the infiltration of macrophages was observed in animals from both sexes of group 4. Black inclusions in the cytoplasm of the infiltrated macrophages were observed in a dose-dependent manner in all animals treated with the test item. Hypertrophy of bronchial and bronchiolar epithelial cells were also observed in animals of both sexes in group 4.

In 4-week recovery animals, the black inclusions in the cytoplasm of the macrophages were still present in all animals treated with the test item. The infiltration of macrophages in group 4 was still observed with higher severity than the controls. The incidence and severity of these findings were comparable between the main study animals and the recovery animals.
The hypertrophy of bronchial and bronchiolar epithelial cells was also observed in animals of both sexes in group 4, but the incidence or severity tended to be decreased after the recovery period.

Table 1 Incidence and Mean Severity Grade of Main Findings in Lungs
Finding
Incidence /
Mean Severity Grade Group 1 (control) Group 2 (0.05 mg/m3) Group 3 (0.25 mg/m3) Group 4 (1.25 mg/m3)
Allocation A
(Main study) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Infiltration of macrophages
Incidence/Mean Severity 5/1.0 4/1.0 5/1.0 5/1.0 5/1.0 5/1.0 5/1.6 5/1.6
Black inclusions in cytoplasm of macrophages
Incidence/Mean Severity 0 0 5/1.0 5/1.0 5/1.8 5/1.8 5/3.0 5/3.0
Hypertrophy, Bronchial/bronchiolar epithelial cells
Incidence/Mean Severity 0 0 0 0 0 0 4/1.3 2/1.5

Allocation B
(4-week recovery) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Infiltration of macrophages
Incidence/Mean Severity 5/1.0 5/1.2 5/1.0 5/1.0 5/1.0 5/1.0 5/1.8 5/1.8
Black inclusions in cytoplasm of macrophages
Incidence/Mean Severity 0 0 5/1.0 5/1.0 5/1.4 5/1.8 5/3.0 5/3.0
Hypertrophy, Bronchial/bronchiolar epithelial cells
Incidence/Mean Severity 0 0 0 0 0 0 2/1.0 2/1.0

Lymph Nodes, Mediastinal
Blackish pigment deposition was observed in the animals of groups 1 and 4 of the allocations A and B. The incidence and severity was not different between them. Based on the shape of this pigment it was considered to be tattoo ink.

Table 2 Incidence and Mean Severity Grade of Main Findings in Lymph Nodes - Mediastinal
Finding
Incidence /
Mean Severity Grade Group 1 Group 2 Group 3 Group 4
Allocation A
(Main study) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Pigment deposition, blackish
Incidence/Mean Severity 1/1.0 3/1.0 - - - - 2/1.0 2/1.0
Allocation B
(4-week recovery) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Pigment deposition, blackish
Incidence/Mean Severity 1/1.0 2/1.0 - - - - 3/1.0 2/1.0


Other Findings
The remainder of findings recorded was the incidental lesions or within the range of normal background lesions which may be recorded in animals of this strain and age.

Description (incidence and severity):

BRONCHO-ALVEOLAR LAVAGE
Biochemical analysis of the broncho-alveolar lavage fluid after the end of treatment resulted in statistically significantly increased GGT levels in group 4 (11.4 vs. 3.2 U/L) and increased protein values for groups 2 to 4. Statistical significance for the latter parameter was obtained for groups 3 and 4 when compared with group 1 (0.95 and 0.83 vs. 0.04 g/L). After the end of the off-treatment period differences to controls diminished and statistical significance was only observed for changes in protein of groups 3 and 4 (0.64 and 0.75 vs. 0.21 g/L).

Differential cell count of the lavage fluid after the end of treatment revealed statistically significantly increased neutrophil levels in group 4 (2.8% vs. 0.3% in controls). After the end of the recovery period the value was similar to controls. During this investigation it was noted that the macrophages in groups 2 to 4 contained phagocytozed material at a dose-related increased incidence. After the end of the recovery the incidence decreased but macrophages with phagocytozed material were still present in all groups.

Dose descriptor:

NOAEC

Effect level:

>= 1.3 mg/m³ air (analytical)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

Exposure to GRAPHISTRENGTH C100 at gravimetrically determined dose levels of 0.066, 0.26 and 1.30 mg/m3 air resulted in findings in the lungs. They consisted of increased severity of the infiltration of macrophages and hypertrophy of bronchial and bronchiolar epithelial cells as well as increased GGT and protein levels in the broncho-alveolar lavage fluid. These findings were noted mainly in high dose animals. Black inclusions in the cytoplasm of infiltrated macrophages in the lungs of all treatment groups correlated well with macrophages with phagocytized material in the broncho-alveolar lavage fluid which was considered to be GRAPHISTRENGTH C100, indicating an adequate exposure of the lungs. Partial recovery was noted after the end of a 4-week treatment free period.

The increase of macrophages correlates to the lung load with the test item and was considered to be related to pulmonary clearance. In the absence of any degenerative and inflammatory lesion in the lungs, these findings were considered to be a normal physiological response and not adverse. The hypertrophy of bronchial/bronchiolar epithelium was deemed to reflect a metabolic reaction. This effect was considered to be adaptive, transient, at a low magnitude, not affecting the overall function of the tissue and this type of reaction is generally considered as reversible (see References (10)). Partial recovery was noted in this study and complete recovery can be assumed at a longer recovery period. Therefore, this finding was also considered to be not adverse.
Decreased kidney weights in males of groups 3 and 4 were considered to be a chance finding in the absence of any histological findings.
On the basis of these findings, a No-Observed-Adverse-Effect-Concentration (NOAEC) of 1.30 mg/m3 air may be established under the conditions of this study.

Executive summary:

In a dose range finding supporting study, Graphistrength™ C100, was administered to three four of 20 male and 10 female Wistar rats by nose-only, flow-past inhalation for a period of five days (6 hours/day) to target concentrations of 0, 0.05, 0.25 and 1.25 mg/m³(Schüler, 2010). A respirable dust aerosol was generated using a SAG 410 Solid Aerosol Generator connected to a micronizing jet mill and a cyclone and an elutriator thereafter. Previously, Graphistrength™ C100 was ground in a ball mill for 20 hours and sieved at a mesh size of 63 µm, in order to facilitate aerosol generation. Throughout the study all animals were observed for viability and clinical signs. In addition, body weight and food consumption were recorded. Subgroups of five males and five females were sacrificed 24 hours after at the last exposure and after a 4-week treatment-free period and subject to a macroscopic examination. Adrenals glands, heart, kidneys, liver, lungs and spleen were weighed and heart, kidneys, larynx, liver, lungs, mediastinal lymph nodes, nasal cavity, naso pharynx, spleen, trachea and all gross lesions were examined microscopically. Additionally, 5 males were also sacrificed at the same time schedule for cytological (cell count, viability and differential cell counts) and biochemical (LDH, ALP, GGT and proteins) examinations of the broncho-alveolar lavage (BAL) fluids. Exposure to gravimetrically determined aerosol concentrations of 0.066, 0.26 and 1.30 mg Graphistrength™ C100 mg/m³air were achieved in groups 2 to 4, respectively. The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined Mass Median Aerodynamic Diameters (MMAD) of 1.93 - 2.56, 1.97 and 1.84 - 1.85 µm and Geometric Standard Deviations (GSD) of 1.30 - 4.33, 1.94 and 1.76 - 1.93, respectively. Mean percentages of particles lower than 3 µm were 68.4, 73.6 and 78.3%, respectively. The generated aerosols were considered to be acceptable for inhalation toxicity testing in rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study. All animals survived to the end of the scheduled treatment period and no clinical signs were observed. There were no test item-related effects on food consumption or body weight. A slight increase in neutrophil count was observed at 1.30 mg/m3 (2.8% vs. 0.3% in controls) after the 5-day treatment, which disappeared after the 4-week recovery period. GGT levels were statistically significantly increased at 1.30 mg/m3 (11.4 vs. 3.2 U/L) but were normal after the recovery and protein values were statistically significantly increased at 0.26 and 1.30 mg/m3 after exposure (0.95 and 0.83 vs. 0.04 g/L) and after the 4-week recovery period (0.64 and 0.75 vs. 0.21 g/L). There were no effects on organ weights or macroscopic findings which were considered to be related to treatment. Treatment-related microscopic findings were noted in the lungs of male and female rats, both after the 5-day exposure and the recovery period. Dose-dependent increased black inclusions were observed in the cytoplasm of infiltrated macrophages, indicating an adequate exposure of the lungs. Macrophage infiltration of the lung (grade 2) was observed at 1.30 mg/m³in 3 males and 3 females after the 5-day exposure and in 4 males and 4 females after the recovery period. Hypertrophy of the bronchial and bronchiolar cells was observed at 1.30 mg/m³in 4 males (3 grade 1 and 1 grade 2) and 2 females (1 grade 1 and 1 grade 2) after the 5-day exposure and in 2 males and 2 females (all grade 1) after the recovery period. No treatment-related microscopic findings were observed in the other organs examined. In conclusion, a 5-day exposure of rats to 1.30 mg/m³of a respirable aerosol of Graphistrength™ C100 induced effects on the lungs, partially reversible after a 4-week treatment free period. On the basis of these findings, a No-Observed-Adverse-Effect-Concentration (NOAEC) of 1.30 mg/m³air may be established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

4.84 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Key study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

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).

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHan™ WIST

Sex:

male/female

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.4 - 3 µm

Geometric standard deviation (GSD):

2.38

Dose / conc.:

0.25 other: mg/m3 air (target conc.)

Remarks:

0.285 mg/m3 (analytical)

Dose / conc.:

1.25 other: mg/m3 air (target conc.)

Remarks:

1.41 mg/m3 (analytical)

Dose / conc.:

5 other: mg/m3 air (target conc.)

Remarks:

5.60 mg/m3 air (analytical)

Control animals:

yes, sham-exposed

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

5.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEC

Remarks:

Local effect

Effect level:

1.41 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

5.6 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

5-day range finding study including a 4 week treatment free recovery period

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at Delivery): 9 weeks
- Body Weight Range (at Acclimatization):
Males: 256.0 to 289.5 g (±8%)
Females: 140.2 to 184.1 g (±15%)
- Fasting period before study: no
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (ad libitum): Pelleted standard Harlan Teklad 2914C
- Water (ad libitum): Community tap-water
- Acclimation period: Fourteen days

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined Mass Median Aerodynamic Diameters (MMAD) of 1.93 - 2.56, 1.97 and 1.84 - 1.85 µm and Geometric Standard Deviations (GSD) of 1.30 - 4.33, 1.94 and 1.76 - 1.93, respectively. Mean percentages of particles lower than 3 µm were 68.4, 73.6 and 78.3%, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only exposure chamber
- Method of holding animals in test chamber: the animals are confined separately in restraint tubes
- System of generating particulates/aerosols:
Prior to use for aerosol generation, the test item was ground in a ball mill for 20 hours. The resulting fine particles were used in the present study. Investigations on the milling process were performed under Harlan Laboratories Study C32562: No surface oxidation (increase of O2 content) was noted after 24 hours of milling and transmission electron microscopy of aerosol filter sampling demonstrated that milling and aerosol generation did not damage the surface of GRAPHISTRENGTH C100.
A dust aerosol was generated from the 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. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer. The aerosol concentrations for groups 2 and 3 were achieved by serial dilution with compressed, filtered, dry air of the aerosol generated for group 4, using an air vacuum device.
- Temperature, humidity, pressure in air chamber:
Aerosol concentration, particle size distribution, relative humidity, temperature and oxygen concentration were measured on test aerosol samples taken at a representative exposure port.
- Air flow rate: flow of air at each tube was between 0.73 to 1.0 L/min
- Method of particle size determination:
The cumulative particle size distribution of the test aerosol was determined using an 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 twice for groups 2 and 4 and once for group 3. The airflow rate through the impactor was approximately 9 L/min. The difference to the lower flow of aerosol air at each tube was considered to be justified in order to increase the impact of the aerosol particles having a low density. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD)
were calculated on the basis of the gravimetric results from the impactor.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
Test aerosol samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The duration of sampling was sufficient to ensure reliable results. Gravimetric determination of the aerosol concentration was performed at twice daily for groups 2 and 3 and once daily for group 4. Sampling flow rate was 3 to 4 L/min per exposure port. During technical trials it was confirmed that the difference to the lower flow of aerosol air at each tube did not affect the results but increased the accuracy of the measurement.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours/day

Dose / conc.:

0.05 other: mg/m3 (target)

Remarks:

0.066 mg/m3 (analytical)

Dose / conc.:

0.25 other: mg/m3 (target)

Remarks:

0.26 mg/m3 (analytical)

Dose / conc.:

1.25 other: mg/m3 (target)

Remarks:

1.30 mg/m3 (analytical)

No. of animals per sex per dose:

20 males and 10 females

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Target concentrations were selected based on published evidence on carbon nanotube
- Rationale for selecting satellite groups:
Allocation and Aerosol Concentrations
mg/m3 air Group 1 Group 2 Group 3 Group 4
control* 0.050 0.25 1.25
0
Males A 1 - 5 21 - 25 41 – 45 61 - 65
B 6 - 10 26 - 30 46 - 50 66 - 70
C 11 – 15 31 – 35 51 – 55 71 – 75
D 16 - 20 36 - 40 56 - 60 76 - 80
Females A 81 – 85 91 – 95 101 – 105 111 – 115
B 86 - 90 96 - 100 106 - 110 116 - 120

* Control animals were treated with air only
A Main study animals
B 4-week recovery animals
C Animals (males) for BAL
D 4-week recovery animals (males) for BAL

- Post-exposure recovery period in satellite groups: 4 weeks

Observations and examinations performed and frequency:

VIABILITY / MORTALITY: Yes
Twice daily during treatment and once daily during acclimatization and recovery.

CAGE SIDE OBSERVATIONS: Yes
Cage-side clinical observations once daily during treatment and recovery. Twice weekly during acclimatization.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Recorded twice weekly (each individual animal) during acclimatization and at days 1 and 5 of the treatment. Weekly during recovery.

FOOD CONSUMPTION:
Recorded twice weekly (per cage) during acclimatization and treatment. Weekly during 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: Yes / No / No data

WATER CONSUMPTION: No:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.

ORGAN WEIGHT: Yes
Adrenal glands, Heart including auricles, Kidneys, Liver, Lungs, instilled via trachea with formalin at approximately 30 cm H2O pressure, Spleen

HISTOPATHOLOGY: Yes
All gross lesions, Heart including auricles, Kidneys, Larynx, Liver, Lungs, Lymph nodes - mediastinal, Nasal cavity, Nasopharyngeal duct and pharynx, Spleen, Trachea - adjacent to larynx and carina and bifurcation

Other examinations:

BRONCHO-ALVEOLAR LAVAGE FLUID (BALF)
5 male animals from each group were sacrificed approximately 24 h after the last (5-day) exposure and after 4 weeks of recovery (Allocation C and D).
Parameters: enzymatic activity of lactate dehydrogenase, alkaline phosphatase, and gamma-Glutamyltransferase, total protein, total cell count, cell viability and differential cell counting (macrophages, neutrophils, lymphocytes, eosinophils, epithelial cells, and other cells).

Statistics:

The following statistical methods were used to analyze the food consumption, body weight, macroscopic findings, organ weights and ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied 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 instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were noted during the course of the study.

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled treatment or recovery periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight which were considered to be related to exposure to the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food intake was similar across all groups during the study.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute kidney weight in males of groups 3 and 4 was statistically significantly reduced as well as the kidney to body weight ratio of group 3. No dose-dependency was noted.
There were no further effects on organ weights which were considered to be possibly related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross lesions that could be attributed to treatment with the test item.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lungs
In the main study animals, increased severity of the infiltration of macrophages was observed in animals from both sexes of group 4. Black inclusions in the cytoplasm of the infiltrated macrophages were observed in a dose-dependent manner in all animals treated with the test item. Hypertrophy of bronchial and bronchiolar epithelial cells were also observed in animals of both sexes in group 4.

In 4-week recovery animals, the black inclusions in the cytoplasm of the macrophages were still present in all animals treated with the test item. The infiltration of macrophages in group 4 was still observed with higher severity than the controls. The incidence and severity of these findings were comparable between the main study animals and the recovery animals.
The hypertrophy of bronchial and bronchiolar epithelial cells was also observed in animals of both sexes in group 4, but the incidence or severity tended to be decreased after the recovery period.

Table 1 Incidence and Mean Severity Grade of Main Findings in Lungs
Finding
Incidence /
Mean Severity Grade Group 1 (control) Group 2 (0.05 mg/m3) Group 3 (0.25 mg/m3) Group 4 (1.25 mg/m3)
Allocation A
(Main study) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Infiltration of macrophages
Incidence/Mean Severity 5/1.0 4/1.0 5/1.0 5/1.0 5/1.0 5/1.0 5/1.6 5/1.6
Black inclusions in cytoplasm of macrophages
Incidence/Mean Severity 0 0 5/1.0 5/1.0 5/1.8 5/1.8 5/3.0 5/3.0
Hypertrophy, Bronchial/bronchiolar epithelial cells
Incidence/Mean Severity 0 0 0 0 0 0 4/1.3 2/1.5

Allocation B
(4-week recovery) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Infiltration of macrophages
Incidence/Mean Severity 5/1.0 5/1.2 5/1.0 5/1.0 5/1.0 5/1.0 5/1.8 5/1.8
Black inclusions in cytoplasm of macrophages
Incidence/Mean Severity 0 0 5/1.0 5/1.0 5/1.4 5/1.8 5/3.0 5/3.0
Hypertrophy, Bronchial/bronchiolar epithelial cells
Incidence/Mean Severity 0 0 0 0 0 0 2/1.0 2/1.0

Lymph Nodes, Mediastinal
Blackish pigment deposition was observed in the animals of groups 1 and 4 of the allocations A and B. The incidence and severity was not different between them. Based on the shape of this pigment it was considered to be tattoo ink.

Table 2 Incidence and Mean Severity Grade of Main Findings in Lymph Nodes - Mediastinal
Finding
Incidence /
Mean Severity Grade Group 1 Group 2 Group 3 Group 4
Allocation A
(Main study) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Pigment deposition, blackish
Incidence/Mean Severity 1/1.0 3/1.0 - - - - 2/1.0 2/1.0
Allocation B
(4-week recovery) 5 M 5 F 5 M 5 F 5 M 5 F 5 M 5 F
Pigment deposition, blackish
Incidence/Mean Severity 1/1.0 2/1.0 - - - - 3/1.0 2/1.0


Other Findings
The remainder of findings recorded was the incidental lesions or within the range of normal background lesions which may be recorded in animals of this strain and age.

Description (incidence and severity):

BRONCHO-ALVEOLAR LAVAGE
Biochemical analysis of the broncho-alveolar lavage fluid after the end of treatment resulted in statistically significantly increased GGT levels in group 4 (11.4 vs. 3.2 U/L) and increased protein values for groups 2 to 4. Statistical significance for the latter parameter was obtained for groups 3 and 4 when compared with group 1 (0.95 and 0.83 vs. 0.04 g/L). After the end of the off-treatment period differences to controls diminished and statistical significance was only observed for changes in protein of groups 3 and 4 (0.64 and 0.75 vs. 0.21 g/L).

Differential cell count of the lavage fluid after the end of treatment revealed statistically significantly increased neutrophil levels in group 4 (2.8% vs. 0.3% in controls). After the end of the recovery period the value was similar to controls. During this investigation it was noted that the macrophages in groups 2 to 4 contained phagocytozed material at a dose-related increased incidence. After the end of the recovery the incidence decreased but macrophages with phagocytozed material were still present in all groups.

Dose descriptor:

NOAEC

Effect level:

>= 1.3 mg/m³ air (analytical)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

Exposure to GRAPHISTRENGTH C100 at gravimetrically determined dose levels of 0.066, 0.26 and 1.30 mg/m3 air resulted in findings in the lungs. They consisted of increased severity of the infiltration of macrophages and hypertrophy of bronchial and bronchiolar epithelial cells as well as increased GGT and protein levels in the broncho-alveolar lavage fluid. These findings were noted mainly in high dose animals. Black inclusions in the cytoplasm of infiltrated macrophages in the lungs of all treatment groups correlated well with macrophages with phagocytized material in the broncho-alveolar lavage fluid which was considered to be GRAPHISTRENGTH C100, indicating an adequate exposure of the lungs. Partial recovery was noted after the end of a 4-week treatment free period.

The increase of macrophages correlates to the lung load with the test item and was considered to be related to pulmonary clearance. In the absence of any degenerative and inflammatory lesion in the lungs, these findings were considered to be a normal physiological response and not adverse. The hypertrophy of bronchial/bronchiolar epithelium was deemed to reflect a metabolic reaction. This effect was considered to be adaptive, transient, at a low magnitude, not affecting the overall function of the tissue and this type of reaction is generally considered as reversible (see References (10)). Partial recovery was noted in this study and complete recovery can be assumed at a longer recovery period. Therefore, this finding was also considered to be not adverse.
Decreased kidney weights in males of groups 3 and 4 were considered to be a chance finding in the absence of any histological findings.
On the basis of these findings, a No-Observed-Adverse-Effect-Concentration (NOAEC) of 1.30 mg/m3 air may be established under the conditions of this study.

Executive summary:

In a dose range finding supporting study, Graphistrength™ C100, was administered to three four of 20 male and 10 female Wistar rats by nose-only, flow-past inhalation for a period of five days (6 hours/day) to target concentrations of 0, 0.05, 0.25 and 1.25 mg/m³(Schüler, 2010). A respirable dust aerosol was generated using a SAG 410 Solid Aerosol Generator connected to a micronizing jet mill and a cyclone and an elutriator thereafter. Previously, Graphistrength™ C100 was ground in a ball mill for 20 hours and sieved at a mesh size of 63 µm, in order to facilitate aerosol generation. Throughout the study all animals were observed for viability and clinical signs. In addition, body weight and food consumption were recorded. Subgroups of five males and five females were sacrificed 24 hours after at the last exposure and after a 4-week treatment-free period and subject to a macroscopic examination. Adrenals glands, heart, kidneys, liver, lungs and spleen were weighed and heart, kidneys, larynx, liver, lungs, mediastinal lymph nodes, nasal cavity, naso pharynx, spleen, trachea and all gross lesions were examined microscopically. Additionally, 5 males were also sacrificed at the same time schedule for cytological (cell count, viability and differential cell counts) and biochemical (LDH, ALP, GGT and proteins) examinations of the broncho-alveolar lavage (BAL) fluids. Exposure to gravimetrically determined aerosol concentrations of 0.066, 0.26 and 1.30 mg Graphistrength™ C100 mg/m³air were achieved in groups 2 to 4, respectively. The particles of the aerosol were considered to be respirable for rats, with gravimetrically determined Mass Median Aerodynamic Diameters (MMAD) of 1.93 - 2.56, 1.97 and 1.84 - 1.85 µm and Geometric Standard Deviations (GSD) of 1.30 - 4.33, 1.94 and 1.76 - 1.93, respectively. Mean percentages of particles lower than 3 µm were 68.4, 73.6 and 78.3%, respectively. The generated aerosols were considered to be acceptable for inhalation toxicity testing in rats and temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study. All animals survived to the end of the scheduled treatment period and no clinical signs were observed. There were no test item-related effects on food consumption or body weight. A slight increase in neutrophil count was observed at 1.30 mg/m3 (2.8% vs. 0.3% in controls) after the 5-day treatment, which disappeared after the 4-week recovery period. GGT levels were statistically significantly increased at 1.30 mg/m3 (11.4 vs. 3.2 U/L) but were normal after the recovery and protein values were statistically significantly increased at 0.26 and 1.30 mg/m3 after exposure (0.95 and 0.83 vs. 0.04 g/L) and after the 4-week recovery period (0.64 and 0.75 vs. 0.21 g/L). There were no effects on organ weights or macroscopic findings which were considered to be related to treatment. Treatment-related microscopic findings were noted in the lungs of male and female rats, both after the 5-day exposure and the recovery period. Dose-dependent increased black inclusions were observed in the cytoplasm of infiltrated macrophages, indicating an adequate exposure of the lungs. Macrophage infiltration of the lung (grade 2) was observed at 1.30 mg/m³in 3 males and 3 females after the 5-day exposure and in 4 males and 4 females after the recovery period. Hypertrophy of the bronchial and bronchiolar cells was observed at 1.30 mg/m³in 4 males (3 grade 1 and 1 grade 2) and 2 females (1 grade 1 and 1 grade 2) after the 5-day exposure and in 2 males and 2 females (all grade 1) after the recovery period. No treatment-related microscopic findings were observed in the other organs examined. In conclusion, a 5-day exposure of rats to 1.30 mg/m³of a respirable aerosol of Graphistrength™ C100 induced effects on the lungs, partially reversible after a 4-week treatment free period. On the basis of these findings, a No-Observed-Adverse-Effect-Concentration (NOAEC) of 1.30 mg/m³air may be established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1.41 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Key study

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The principal health findings of a sub-chronic inhalation exposure to high concentrations of Graphistrength C100 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 [Pauluhn J, 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 (2010) 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 and not adverse response to the overload of the lung with insoluble particles (Pauluhn J, 2010).

Justification for classification or non-classification

According to the available data and Regulation (EC) No 1272/2008, no classification of Graphistrength C100 is required for repeated dose toxicity.