[No public or meaningful name is available]

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jan 2010 - 13 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Vapour grown graphitic carbon fibre

Molecular formula:

C

IUPAC Name:

Vapour grown graphitic carbon fibre

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, North Carolina, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: 241.8 - 251.3 g (males), 176.8 - 182.8 g (females)
- Housing: 2 rats per cage (sexes separated) in solid-bottom polycarbonate cages with nestlets for enrichment
- Diet: PMT Nutrition International, LLC, Certified Rodent LabDiet 5002, ad libitum, except during exposures and during the urine collection period.
- Water: tap water, ad libitum, except during exposures and during the urine collection period.
- Acclimation period: 6 days quarantine

DETAILS OF FOOD AND WATER QUALITY:
Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The
presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 23 March To: 20 Sept 2010

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.9 - <= 3.3 µm

Geometric standard deviation (GSD):

3.1

Remarks on MMAD:

Rats from the 0.50 mg/m³ low target exposure were exposed to an aerosol concentration of 0.54 ± 0.018 mg/m³ (mean ± SEM), which was characterized by MMAD of 1.9 µm (3.1 GSD) and a fiber count of 4.9 ± 3.5 fibers/cm³ (mean ± SD). Rats from the 2.5 mg/m³ intermediate target level were exposed to an aerosol concentration of 2.5 ± 0.039 mg/m³, which was characterized by a MMAD of 3.2 µm (2.1 GSD) and a fiber count of 56.0 ± 30.6 fibers/cm³. Rats from the 25 mg/m³ high target level were exposed to an aerosol concentration of 25 ± 0.3 mg/m³, which was characterized by a MMAD of 3.3 µm (2.0 GSD) and a fiber count of 52 ± 143.2 fibers/cm³.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Constructed of stainless steel and glass; nominal internal volume: 150 L. Uniform distribution of test atmosphere promoted by tangential feed and stainless steel baffle at the chamber inlet.
- Method of holding animals in test chamber: Perforated stainless steel cylinders with conical nose pieces
- System of generating particulates/aerosols: Test substance was metered into a glass tube using an Accurate model 102M or 106M bin feeder. A filtered, high-pressure air stream carried the resulting atmosphere into the exposure chamber. A diluation air stream of filtered, high pressure air entered the chamber near the turret.
- Temperature, humidity, oxygen concentration in air chamber: 20 - 24 °C, 30 - 70%, at least 19%
- Air change rate: at least 10 /h
- Method of particle size determination: Sierra series 210 cyclone preseparator/cascade impactor and Sierra series 110 constant flow air sampler.
- Treatment of exhaust air: Through a high-capacity canister filter prior to discharge into the fume hood.

TEST ATMOSPHERE
- Brief description of analytical method used:
Concentration of test substance were determined by gravimetric analysis. Known volumes of chamber atmosphere were drawn from the sampling port through a 25 mm filter cassette containing a pre-weighed glass fiber filter. Atmospheric concentration of the test substance was calculated from the difference between the pre- and post-sampling filter weights divided by the volume of chamber atmosphere sampled.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test substance were determined by gravimetric analysis.

Duration of treatment / exposure:

13 weeks and approx. 3 months post-exposure recovery period (satellite control and high concentration test groups), a total of 65 exposures

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.54 mg/m³ air (analytical)

Remarks:

MMAD = 1.9 µm (GSD = 3.1), fiber count = 4.9 ± 3.5 fibers/cm³

Doses / concentrations 2

Dose / conc.:

2.5 mg/m³ air (analytical)

Remarks:

MMAD = 3.2 µm (GSD = 2.1), fiber count = 56.0 ± 30.6 fibers/cm³

Doses / concentrations 3

Dose / conc.:

25 mg/m³ air (analytical)

Remarks:

MMAD = 3.3 µm (GSD = 2.0), fiber count = 252 ± 143.2 fibers/cm³

No. of animals per sex per dose:

10 (main study)
5 (BAL investigation, main study)
10 (recovery study, control and high-concentration groups)
5 (BAL investigation, recovery study, control and high-concentration groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Concentrations were based on a previous one-week pilot study
- Post-exposure recovery period in satellite groups: approx. 3 months

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 0 and weekly thereafter
- Parameters checked/examined: fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and weekly thereafter

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Week 13, and Week 25
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of exposure period, Day 92, for all exposure groups, and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (at least 15 h)
- How many animals: 10 males and 10 females from each exposure group
- Parameters checked/examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular (cell) volume, mean corpuscular (cell) hemoglobin, mean corpuscular (cell) hemoglobin concentration, prothrombin time, activated partial thromboplastin time, red cell distribution width, absolute reticulocyte count, platelet count, white blood cell count, differential white blood cell count, microscopic blood smear examination, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of exposure period, Day 92, for all exposure groups, and at the end of the recovery period
- Animals fasted: Yes (at least 15 h)
- How many animals: 10 males and 10 females from each exposure group
- Parameters checked/examined: aspartate aminotransferase, alanine aminotransferase, total protein, albumin, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, creatinine, cholesterol, triglycerides, glucose, globulin, calcium, inorganic phosphorus, sodium, potassium, chloride, bile acids, C-reactive protein

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of exposure period, Day 92, for all exposure groups, and at the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (at least 15 h)
- Parameters checked/examined: quality, color, clarity, volume, specific gravity, pH, glucose, ketone, bilirubin, blood, urobilinogen, protein, microscopic urine sediment

OTHER:
- Bronchoalveolar lavage (BAL): Yes
- Pulmonary cell proliferation assessment: Yes

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes (liver, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, pancreas, kidneys, urinary bladder, lungs, trachea, nose (4 cross sections), larynx, pharynx, heart, aorta, spleen, thymus, mandibular lymph node, mesenteric lymph node, bone marrow, Peyer's patches, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain (including cerebrum, cerebellum, medulla/pons), spinal cord (cervical, mid-thoracic, lumbar), sciatic nerve, eyes, optic nerves, skeletal muscle, femur/knee joint, sternum, testes, epididymides, prostate, seminal vesicles, ovaries (including oviducts), uterus (including cervix), mammary glands, vagina, skin)

Statistics:

Refer to 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic observations of hair loss, stained fur and/or superficial wounds were recorded across all exposure groups. These were not considered test substance­related or adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

0.54 mg/m³: One male was found dead in its cage on Day 41. The death was considered spurious and not related to test substance exposure.
2.5 mg/m³: One male was accidentally killed while receiving the intraperitoneal injection of BrdU on test Day 92.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

0.54 mg/m³: Males exhibited lower mean body weights than the control group starting on Day 14 until the end of the exposure period associated with sporadic reductions in body weight gains and reduced food consumption.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

0.54 mg/m³: Males showed statistically reduced food consumption during the 90-day exposure period and sporadic reductions in food efficiency which was associated with body weight reductions.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

25 mg/m³: One female showed a detached retina on test Day 79. The finding was considered to be spurious and unrelated to test substance exposure.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

25 mg/m³: Mean absolute neutrophil count (ANEU) was increased in males and females, and mean eosinophil count (AEOS) was increased in females (non adverse).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male rats of all exposure levels (0.54, 2.5 and 25 mg/m³): Mean triglycerides (TRIG) were slightly lower when compared to controls.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

25 mg/m³ (males and females) and 2.5 mg/m³ (females): increases in lung weights

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

0.54, 2.5, 25 mg/m³: Concentration-related incidence of fiber-laden alveolar macrophages. Fibers in the nasal turbinate sections and in other levels of the nose.
2.5, 25 mg/m³: Concentration-related centriacinar subacute/chronic inflammation in the lungs with infiltrates of inflammatory cells.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2.5, 25 mg/m³: Thickening of interstitial walls and hypertrophy/hyperplasia of type II pneumocytes.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

2.5, 25 mg/m³: Extrapulmonary fibers were observed in the following organs/tissues: glomeruli of the kidneys, heart, brain (choroid plexus), duodenum, ileum, liver (Kupffer cells), spleen, mediastinal lymph node.

Details on results:

IN-LIFE MEASUREMENTS
BODY WEIGHTS AND BODY WEIGHT GAINS
0.54 mg/m³: Males demonstrated lower mean body weights starting on test day 14 and lasted until the end of the exposure period. Lower mean body weights were associated with sporadic reductions in body weight gains and reduced food consumption. There was no concentration-response relationship. The findings are non-adverse and are not considered test substance-related.
25 mg/m³: During the recovery period, females demonstrated statistically increased body weight gains.

FOOD CONSUMPTION
0.54 mg/m³: Males demonstrated statistically reduced food consumption and sporadic reductions in food efficiencies.
2.5 mg/m³: Females demonstrated transient increases in food consumption and no changes in food efficiency.
25 mg/m³: Females showed an increase in food consumption. Males demonstrated transient reductions in food consumption and in food efficiency. Transient increases and decreases in food efficiency were also observed in females.
All of the observed differences in food consumption and food efficiency in males and females were transient in nature and did not exhibit concentration-response relationship and were not considered test item-related.

CLINICAL OBSERVATIONS AND MORTALITY
0.54 mg/m³: One male rat was found dead in its cage on the morning of test day 41. Gross histopathological evaluation revealed dark foci of the thymus, corroborating with the microscopic finding of grade 3 hemorrhage. Other microscopic findings included grade 2-3 congestion of the adrenal glands, kidneys, liver, mediastinal lymph node, lung and spleen. The findings would be expected in an animal that was found dead. Therefore, the death was considered spurious and not to test item related.
2.5 mg/m³: One male was accidentally killed while receiving the intraperitoneal injection of BrdU on test day 92.

There were no adverse clinical observations in any of the exposure groups during the 6 h exposures, immediately following the exposures, at cage-site evaluations or in detailed clinical observations. Sporadic observations of hair loss, stained fur and/or superficial wounds were recorded across all exposure groups and were not considered test item-related or adverse.

OPHTHALMOLOGY EVALUATION
25 mg/m³: One female demonstrated a detached retina on test day 79. No other rats exposed to the test item demonstrated abnormal findings upon ophthalmological examination. The retinal detachment in the single female animal is, therefore, considered to be spurious and unrelated to test substance exposure.

CLINICAL PATHOLOGY EVALUATION
HEMATOLOGY
There were no adverse changes in group mean hematology parameters in male or female rats at test day 92. The following changes in mean hematology parameters were related to exposure to the test item but considered non-adverse:
25 mg/m³: Mean absolute neutrophil count (ANEU) was increasedI in males and females (28% and 70% above controls, respectively) and mean eosinophil count (AEOS) was increased in females (56% above control). Other changes in group means for ANEU and AEOS were minimal. Nevertheless, these minimal increases in leukocyte parameters were likely correlative to inflammatory changes in the lungs observed histologically and thus, were considered to be related to exposure to the test material. Following the 3-month recovery, group mean values for most white cell parameters were similar to controls except ANEU in females (120% above the control), which remained elevated (primarily due to one animal).

The following changes in mean hematology parameters were considered to be unrelated to treatment and, thus, non-adverse:
25 mg/m³: Mean platelet count (PLT) was minimally higher (12% above the control) in males. The difference was not dose-related across concentration groups and, with few exceptions, individual PLT values were similar across the control and treated groups. No changes in PLT were observed in females at any exposure concentration.

The following changes in mean coagulation parameters were not adverse or not related to exposure to the test idem:
25 mg/m³: Mean prothrombin time (PT) was minimally shortened (8% below the control) in males. With the exception of 2 animals, the range of individual values of PT was similar to control males. Additionally, decreases in coagulation times have no toxicological significance. Therefore, this change was considered to be unrelated to treatment and non-adverse.

CLINICAL CHEMISTRY
There were no treatment-related changes in group mean clinical chemistry parameters in male or female rats. The following changes in mean clinical chemistry parameters were not adverse or not related to exposure to the test substance:
0.5, 2.5 and 25 mg/m³: Mean triglycerides (TRIG) were slightly lower in males (40%, 31% and 21% below the control, respectively). These differences did not occur in a dose-related manner and most individual TRIG values were within or slightly below the historical control range for similarly aged males. There were no changes in mean cholesterol values for males nor were there changes in female cholesterol or triglycerides. Therefore, this change was not considered to be test item-related.

The following changes in group mean clinical chemistry parameters at test day 181 (males) and 180 (females) were considered to be unrelated to treatment and non­adverse because the change occurred only after 3-months of recovery:
25 mg/m³: Mean C-reactive protein (C-RP) was minimally decreased in males (10% below the control) and mean total bile acids (TBA) was minimally increased in females (106% above the control).

ANATOMIC PATHOLOGY EVALUATION
ORGAN WEIGHTS
Test item-related increases in lung weights were present in males and females in the 25 mg/m³ and in female rats in the 2.5 mg/m³ exposure groups. Mean absolute lung weight was increased 22% and 38% above control in the 25 mg/m³ group, respectively. ln the 2.5 mg/m³ group, mean absolute lung weight was increased 15% above control. Lung weight changes were partially, but not completely, reversible following the one-month recovery period, as mean absolute lung weight was 16% and 21% higher than control in the 25 mg/m³ male and female recovery groups, respectively.

There were no other organ weight changes considered to be related to exposure to the test material. A number of organ weight differences were observed but were not considered exposure-related. These included the following fndings:
Lung weight relative to body weight was higher in the 0.5 and 2.5 mg/m³ male groups. However, these differences did not occur in a concentration-related manner and were not associated with statistically significant changes in other lung weight parameters. Especially in the 0.5 mg/m³ group, these changes were primarily the result of lower group mean terminal body weight. Group mean thymus weight relative to body weight was higher in all exposed male groups. Absolute thymus weight and thymus weight relative to brain weight was also higher in the 2.5 mg/m³ male group. These differences did not occur in a concentration-related manner and were not associated with microscopic changes in the thymus. Therefore, these thymus weight changes were considered to be unrelated to exposure to the test item. Lower terminal body weight in the 0.5 mg/m³ male group resulted in statistically significant differences in one or more organ weight parameters in several organs including adrenal glands, brain, spleen, and epididymides. These differences were not concentration-related, were not associated with correlative microscopic findings and, thus, were not considered to be primary effects of exposure to the test item. Absolute spleen weight relative to body weight was lower in 25 mg/m³ females at the end of the exposure period. This difference was not associated with changes in other spleen weight parameters or with microscopic changes in the spleen and was considered to be unrelated to exposure to the test item. All other organ weight differences were considered spurious and unrelated to exposure to the test item because they did not occur in a concentration­related manner and/or were only seen in recovery groups.

TERMINAL SACRIFICE
Exposure-related findings in the lungs were noted in all exposed rats at all groups. These findings consisted of a concentration-related incidence of fiber-laden alveolar macrophages in all treated groups and concentration-related centriacinar subacute/chronic inflammation in the mid­ and high-concentration lungs. The fibers within the alveolar macrophages formed black tangles which were birefringent. These macrophages were located in the terminal bronchiole and alveolar duct areas of the lung. Fibers were also sometimes observed in interstitial or bronchial-associated lymphoid tissue (BALT), but were not diagnosed separately. Fiber-laden alveolar macrophages were present in all treated rats at this interval and were graded minimal in the low-concentration, slight in the mid-concentration, and moderate in the high-concentration.
The inflammation in the lungs was located in the terminal bronchiole and alveolar duct areas (centriacinar) in areas in which the fiber-laden alveolar macrophages had accumulated. This finding consisted of infiltrates of inflammatory cells with some thickening of interstitial walls and hypertrophy/hyperplasia of type II pneumocytes. This type of inflammation was noted in all 10 of the high-concentration rats of each sex and was graded slight and in 7 rats of each sex in the mid-concentration group where it was graded minimal. It was not noted in the low­concentration or control rats.
Exposure-related findings were present in the nasal passages of treated rats. Fibers were also noted, individually and in tangled bundles, in the nasal turbinate sections of all groups of test item exposed rats. Fibers were present in the first level of the nose in all 10 males and females in the high-concentration, in 8 males and 9 females in the mid-concentration and in 3 rats of each sex in the low-concentration exposure groups. Fibers were also noted occasionally in other levels of the nose. Additionally, eosinophilic hyaline droplets were noted in the mucosal epithelium of the nasal turbinates, especially levels II, III, and IV. The incidence and/or severity were exacerbated in an exposure-dependent manner. This finding is considered to be a non-specific adaptive or protective response to prolonged inhalation of irritants. Acute to subacute inflammation, characterized by infiltration of neutrophils or mixed inflammatory cells, was also present in a concntration-related manner in the submucosa of the nasal turbinates.
Extrapulmonary fibers were also noted in the glomeruli of the kidneys in all mid- and high­concntration rats and in 8 males and 5 females at the lowest exposure level and in the heart of 2 males and one female at the highest level of exposure. In the tissues that were only examined from control and high-concentration rats, fibers were noted in rats at the highest exposure level in the brain (choroid plexus) of 2 males and 2 females, in the duodenum of one male and one female, in the ileum of one female, in the liver (Kupffer cells) of 9 males and 10 females, and in the spleen of 10 males and 7 females. Fibers were also noted in the mediastinal lymphnode (not a protocol-required tissue) in 5 of 6 males and one of 2 females. No reactions were noted in the tissues containing these fibers. A variety of spontaneous disease lesions and incidental findings were noted in various tissues without respect to treatment. These findings were the usual type and number commonly observed in rats of this age and strain.

RECOVERY SACRIFICE
After the three month recovery period fiber-laden alveolar macrophages were still noted in the terminal bronchiole areas of the lungs in all ten males and females at the highest exposure level. There was essentially no change in the severity grade for this finding that had been noted at the terminal sacrifice. The subacute/chronic inflammation noted in the centriacinar areas of the lung was less severe than was noted at the terminal sacrifice. At terminal sacrifice, all of the highest exposure level rats of both sexes had inflammation grades with a mean grade of 2.0, but after recovery, the mean grade was 1.8 for the males (10/10 affected) and 1.1 for the females (8/10 affected). A few fiber-laden alveolar macrophages were also noted focally in the lungs of one control male, but in the alveoli and not the area noted in the treated animals. This finding was most likely contamination that occurred during processing.
In the nose, fibers were still present in all of the rats of both sexes at the highest exposure level after the recovery period. However, the incidence of inflammation was markedly reduced and the presence of eosinophilic hyaline droplets was somewhat reduced in degree.
Occasional individual fibers, without evidence of a tissue response, were noted in the glomeruli of the kidneys of all rats at the highest level of exposure after the recovery period.
A variety of spontaneous disease lesions and incidental findings were noted in the limited tissues examined at this sacrifice interval. lncluded among these findings was a single female in the high-concentration was diagnosed with histiocytic sarcoma in the heart, liver, lungs, mandibular lymph node, and skin. These findings were the usual number and type commonly noted in rats of this age and strain.

BRONCHOALVEOLAR LAVAGE (BAL)
PULMONARY INFLAMMATION
The numbers of cells recovered by BAL from exposed rats were not significantly higher than any of the other groups for the 90-day exposure period or at 3 months post 90-day exposure for the control and high dose groups.
Inhalation exposures to the test item produced small but significantly increased neutrophilic inflammation in both male and female rats at 25 mg/m³ exposures (ca. 11-12% PMNs). The inflammatory response in high dose exposed males was still relatively minor, but significantly increased vs. air controls at 3 months post exposure for male rats, indicating that the inflammatory response measured at 90 days had not been resolved at 90 days post exposure.

BAL FLUID LDH, ALKP, MICROPROTEIN AND PHAGOCYTIC MACROPHAGES
25 mg/m³: Increases in BAL fluid Lactate Dehydrogenase (LDH), Alkaline phosphatase (ALKP), and Microprotein (MTP) values in males and females both at 90 days exposure and following 3 months post exposure.
0.54 mg/m³: > 60% of pulmonary macrophages contained test item (males and females).
2.5 and 25 mg/m³: > 90% of macrophages contained test item. After the 3-month recovery period, > 70% of macrophages from the 25 mg/m³ group still contained test item.

PULMONARY CELL PROLIFERATION STUDIES
TERMINAL BRONCHIOLAR CELL PROLIFERATION RATES
25 mg/m³: Increases in airway cell proliferation were measured in males and females one day following final expousre. Moreover, the increase in cell proliferation in the terminal bronchiolar cells had reversed following the 3 month recovery period.

LUNG PARENCHYMAL CELL PROLIFERATION RATES
25 mg/m³: Increased lung parenchymal cell proliferation rates were measured in males and females following 90 days of exposure. During the 3-month post exposure period, cell proliferation rates in BrdU cell labeling indices were significantly increased in males.

SUBPLEURAL/MESOTHELIAL CELL PROLIFERATION RATES
25 mg/m³: Increased subpleural/mesothelial cell proliferation rates were measured in males and females following 90 days of exposure. Most of the increased labeling of immunostained cells was associated with cells located in distal alveolar ducts adjacent or sub-adjacent to the visceral pleura. Positive staining cells were adjacent to macrophages containing test item particulates in distal lung sites. During the 3-month recovery period, cell proliferation rates in BrdU cell labeling indices were increased in female but not male rats. Similar to the assessments made at the 90-day exposure period, the enhanced numbers of labeled cells in female rats (0.98% vs.0.74% labeled cells for the 25 mg/m³ and control groups, respectively) were anatomically located in subpleural sites.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

0.54 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at 0.54 mg/m³

2

Dose descriptor:

LOAEC

Effect level:

2.5 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: elevated BAL recoveries of PMNs, LDH, ALKP and MTP and increased cell proliferation at 25 mg/m³

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Exposure of male and female Sprague-Dawley rats to concentrations of 0.54, 2.5 and 25 mg/m3 of test item resulted in a concentration-related accumulation of fibers within alveolar macrophages and, at the two highest exposure concentrations, a subacute to chronic inflammation of the terminal bronchiole and alveolar duct areas of the lungs. After a three month recovery period, the fiber-laden alveolar macrophages still persisted in the lungs. However, the centriacinar inflammation was less severe.

Fibers were also noted in the nasal turbinates (particularly level I) in all rats exposed to the highest concentration, nearly all of the rats at the mid-level, and a few rats at the lowest concentration. At the highest exposure level, this finding also persisted after the three-month recovery period. A non-specific reaction to exposure of the nasal passages resulted in the appearance of eosinophilic hyaline droplets in the nasal mucosa and a minimal to mild inflammatory response in the submucosa. After the three-month recovery, the eosinophilic hyaline droplets persisted, but were somewhat decreased, and the inflammatory response was minimal.

The No-Observed-Adverse-Effect-Level (NOAEL) for the test item is considered to be 0.54 mg/m3 (corresponding to 4.9 fibers/cm3) for male and female rats. This NOAEL is based on the slight subacute to chronic inflammation of the terminal bronchiole and alveolar duct areas of the lungs with some thickening of interstitial walls and hypertrophy/hyperplasia of type II pneumocytes in rats exposed to 2.5 mg/m3 and the minimal inlammation of the terminal bronchiole and alveolar duct areas in rats exposed to 25 mg/m3 test item as well as the elevated BAL recoveries of PMNs, LDH, ALKP and MTP and increased cell proliferation observed in animals exposed to 25 mg/m3.