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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant non-guideline study with well-characterized test material

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
5-day inhalation exposure with 21 days recovery group.
Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Extractable fraction 0.5 %
- Remaining solvent < 10 ppm
- Homogeneity: homogeneous
- Date of production: date of filling 30 Jan 2012
- Physical state/ Appearance: Solid / red
- Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: Main group day 0: control group: 229.0 - 269.7g; test group: 228.5 - 264.9g, Recovery group day 0: control group: 209.9 - 264.1g; test group: 234.1 - 276.5g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Mean relative humidities in the inhalation systems ranged between 40.4 and 52.9 %. Mean temperatures in the inhalation systems ranged between 21.3 and 22.4°C. They are within the range suggested by the respective testing guidelines.

Administration / exposure

Route of administration:
other: dust aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The two measurements of particle size resulted in MMADs 0.6 and 1.1 μm with GSDs 2.5 and 3.2, respectively. The calculated mass fractions of particles below MMAD 3 μm was 87.1 and 90.1 %.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The concentration was adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
- The following test substance flow, air flows and evaporation temperatures were scheduled: Test group; Substance flow (g/h); Supply air 1 conditioned (m³/h); Supply air 2 compressed (m³/h); Exhaust air (m³/h)
0; -; 6.0 ± 0.3; -; 5.7 ± 0.3
1; 0.10 – 0.25; 4.5 ± 0.3; 1.5 ± 0.3; 5.4 ± 0.3

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems (test group 1 in INA 60, volume V ≈ 90 L, BASF SE, control group in INA 120, volume V ≈ 120 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
- Exposures: The head-nose exposure technique was preferably selected for this aerosol/dust/ inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.

VEHICLE
- Composition of vehicle: Conditioned supply air is activated charcoal filtered air conditioned to about 30% - 70% relative humidity and 20°C - 24°C. Compressed air is filtered air pressurized to about 6 bar.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CALCULATION OF NOMINAL CONCENTRATIONS: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
ANALYTICAL DETERMINATION OF CONCENTRATIONS: The concentration of the inhalation atmosphere in test group 1 was analyzed by gravimetry. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In the test group, the constancy of the dust atmosphere in the chamber was continuously monitored using scattered light photometers. The analyses were carried out at the Laboratory for Inhalation Toxicity of the Experimental Toxicology and Ecology of BASF SE.
- Sampling for gravimetric analyses: Equipment: Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA), Internal probe diameter: 7 mm, Filter: MN 85/90 BF (d = 4.7 cm), Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA), Balance: Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany). Sampling: Sampling velocity: 1.25 m/s, Flow rate of sampling: 3 L/min, Sample volumes Test group 1: 90 L, Sampling site: immediately adjacent to the animals' noses at a separate spare port, Sampling frequency: as a rule, 3 samples per exposure and concentration group
- Gravimetrical method of analyses: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
REAL TIME MONOTORING OF CONSTANCY OF CONCENTRATIONS: Scattered light photometer (VisGuard (Sigrist) in test group 1 was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.
PARTICLE SIZE ANALYSIS:
- Definitions: EACD 50%(effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages. MMAD(mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass. Geometrical standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.
- Equipment, sampling and method of determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA), Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA), Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA), Sampling probe internal diameter 7 mm, Balance Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany), Mettler XP 205.(Mettler-Teldo AG, Greifensee, Switzerland)
- Sampling for particle size analyses: Preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and 2 samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals. A sample volume of 90 L was used in each test group. Method of analysis: Gravimetrical determination. The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting disc and backup filter before and after sampling. The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter. Evaluation: The calculation of the particle size distribution was carried out in the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen and DIN 66161: Partikelgrößenanalyse, Beuth-Vertrieb GmbH, Berlin und Köln, Germany).
- Particle size distribution measurements with APS: Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used (TSI APS 3321). Frequency: on two days during the exposure period, with 3 repeats on each day.
- Particle size distribution measurements with Optical particle counter: For each test atmosphere measurements with an optical particle counter (WELAS 2000; (Palas® GmbH, Karlsruhe, Germany)) were performed to determine the size distribution of particles with diameters larger than 246 nm. The WELAS 2000 uses a white-light source to illuminate a measurement volume through which particles have to move singly. The measuring range of the sensor was 0.246 to 9.653 μm and the sampling flow rate 5 L/min.
- Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 μm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.083 μm according to the software calculation. The duration of each measurement cycle was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
daily, for five consecutive days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
29.86 +/- 2.77 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)
Control animals:
yes, concurrent vehicle
Details on study design:
On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Examinations

Observations and examinations performed and frequency:
MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

CLINICAL OBSERVATIONS: The clinical condition of the test animals was recorded once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).
ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25A2M). Rat haptoglobin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25HPT). Both ELISA kits were measured with a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer.

BRONCHOALVEOLAR LAVAGE FLUID (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs at a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA produced by Bender MedSystems, Vienna, Austria (cat. no BMS631INST), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA produced by R&D Systems Inc., Minneapolis, US, (Quantikine rat CINC-1, cat. no. RCN100), Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA produced by R&D Systems Inc., Minneapolis, USA (cat no. MMC00), Rodent osteopontin measured with an ELISA produced by R&D Systems, Inc., Minneapolis, US (Quantikine mouse osteopontin, cat. no. MOST00).
Sacrifice and pathology:
NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve and eyelids, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy. Tissues and organs to be examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).
Statistics:
- Body weight, body weight change: A comparison of the dose group with the control group was performed using the student t-test (two-sided) for the hypothesis of equal means.
- Clinical Pathology parameters in Blood: Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (two-sided) for the equal medians.
- Clinical Pathology parameter in BALF, Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (one-sided) for the
equal medians.

Throughout the sections of the report, when intergroup differences are referred to as "significant " it implies that the differences have attained statistical significance (p ≤ 0.05) when compared with the control group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
orange red discoloration by pigment
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Reversible minimal hypertrophy/hyperplasia in the bronchioles, at the level of the terminal bronchioles and alveolar ducts. Clearance of pigment deposits by macrophages
Details on results:
CLINICAL SIGNS AND MORTALITY: During the pre- and post-exposure period the animals showed no clinical signs and findings different from normal. During the exposure period, substance-contaminated fur was observed after the daily exposure. No deaths were recorded throughout the study.

BODY WEIGHT AND WEIGHT GAIN: The mean body weights of the test substance exposed group (test group 1) were not statistically significantly different from the control (test group 0) during the whole study. The body weight change of the exposed animals was statistically not different from the controls.

HAEMATOLOGY: No treatment-related, adverse changes among hematological parameters were observed.

CLINICAL CHEMISTRY: No treatment-related, adverse changes among clinical chemistry parameters were observed.

ACUTE PHASE PROTEINS IN SERUM: No treatment-related changes of the acute phase protein levels in serum were observed.

BRONCHOALVEOLAR LAVAGE FLUID (BAL): (see tables 2)
- Cytology in BAL: Not treatment-related, adverse changes among cytological BAL parameters were observed.
- Total proteins an enymes in BAL: Not treatment-related, adverse changes among protein and enyzyme parameters were observed.
- Antigens in BAL: No treatment-related changes among antigen levels in BAL were observed.

ORGAN WEIGHTS:
The absolute and relative weight parameters showed no relevant changes and are considered to be within the normal range.

MACROSCOPIC CHANGES
An orange red discoloration was observed in the lungs of all treated males and in the fur of most of them. This finding is regarded as treatment-related and was due to the presence of the test substance. While the fur discoloration is not considered adverse, the lung discoloration was associated with histopathological findings. No other macroscopic changes were noted.

PATHOLOGY
Main group (end of treatment) (see table 1):
The test substance was mainly stored in the cytoplasm of macrophages as brownish-orange fine pigment particles. A moderate number of pigment-laden macrophages were diffusely distributed in the lungs but were predominantly present in the lumen of terminal bronchioles, alveolar ducts and adjacent alveoli. The numberof pigment particles in the macrophages was also moderate. The epithelial hypertrophy and/or hyperplasia was generally observed at the level of terminal bronchioles and alveolar ducts and in more proximal located bronchioles. This finding was characterized by an increased size and number of bronchiolar epithelial cells, accompanied by a slight cytoplasmic basophilia.
In the nasal cavity and larynx, minimal to slight amounts of free pigment particles were found admixed with the mucus secretion.
Minimal focal changes in the larynx, such as epithelial alteration in the ventral pouch of the epiglottis or lymphoid infiltrations were observed, but are considered to be background lesions that are not related to treatment. No findings were noted in the trachea.
Minimal number of pigment-laden macrophages with minimal amount of pigment particles was observed in the mediastinal and tracheobronchial lymph nodes.

Recovery group (see table 1)
Compared with the control group 0, all males of the test group showed slight number of pigment-laden macrophages diffusely distributed in the lung with a marked tendency to accumulate but not to aggregate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). The bronchus-associated lymphoid tissue (BALT) of all males showed minimal number of pigment-laden macrophages.
minimal amounts of free pigment particles were found in the lumen of the nasal cavity in 1 of 3 males. All other findings in the nasal cavity and larynx (lymphoid infiltrations and epithelial alterations) were of minimal magnitude and occurred in 1 of 3 animals. All of them are regarded as background lesions without relation to treatment. No findings were observed in the trachea.
A minimal to slight number of pigment-laden macrophages was found in the tracheobronchial lymph node

Effect levels

Dose descriptor:
conc. level:
Effect level:
29.86 mg/m³ air
Based on:
test mat.
Remarks:
mean measured concentration
Sex:
male
Basis for effect level:
other: Reversible minimal hypertrophy/hyperplasia in the bronchioles, at the level of the terminal bronchioles and alveolar ducts. Clearance of pigment deposits by macrophages.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

In general the generated aerosols of the test substances were within the respirable range. APS 3321 showed MMADs which are higher than the measured values by cascade impactor. This difference may be attributed to bouncing-off effect of the particles within the cascade impactor. However, light refraction of the strong colored pigment particles may also interfere the measurement by APS 3321. Although the APS measurements indicated some large particles, the data of WELAS and SMPS indicate majority of particles < than 1 μm. Figures are shown in the attached pdf file.

Table 1: Histopathology findings in lung immediately after treatment and after recovery

    main group recovery group
  control group test substance 30 mg/m3 test substance 30 mg/m3
Animals examined 3 3 3
Macrophages, pigment-laden 0 3 3
·   Grade 2     3
·   Grade 3   3  
Macrophages, pigment-laden, in BALT (bronchus-associated lymphoid tissue) 0 0  
·   Grade 1     3
Free pigment particles 0 0 0
Hypertrophy/hyperplasia, terminal bronchiole 0 3  
·   Grade 1   3  

Tables 2 Broncheoalveolar lavage (days 7 - 28)

BAL: cytology
Air control; males

30 mg/m3
males
TCN BAL Mean 82.59 x+ 67.30
[cn/µL BAL] S.d. 17.82 11.36
day 7 N 5 5
  Median 75.00 68.89
MPH BAL_C Mean 79.77 x+ 60.97
[cn/µL BAL] S.d. 17.90 8.20
day 7 N 5 5
  Median 73.69 60.00
LY BAL_C Mean 0.29 x+ 0.26
[cn/µL BAL] S.d. 0.22 0.30
day 7 N 5 5
  Median 0.26 0.17
PMN BAL_C Mean 2.34 x+ 6.03
[cn/µL BAL] S.d. 1.28 5.17
day 7 N 5 5
  Median 1.86 5.49
MO BAL_C Mean 0.00 x+ 0.00
[cn/µL BAL] S.d. 0.00 0.00
day 7 N 5 5
  Median 0.00 0.00
EO BAL_C Mean 0.19 x+ 0.00
[cn/µL BAL] S.d. 0.21 0.00
day 7 N 5 5
  Median 0.17 0.00
ATY BAL_C Mean 0.00 x+ 0.04
[cn/µL BAL] S.d. 0.00 0.09
day 7 N 5 5
  Median 0.00 0.00
MPH BAL% Mean 96.45 x+ 91.20
[%] S.d. 1.89 7.12
day 7 N 5 5
  Median 96.25 91.25
LY BAL% Mean 0.35 x+ 0.35
[%] S.d. 0.29 0.42
day 7 N 5 5
  Median 0.25 0.25
PMN BAL% Mean 2.95 x+ 8.40
[%] S.d. 1.74 6.95
day 7 N 5 5
  Median 2.75 7.75
MO BAL% Mean 0.00 x+ 0.00
[%] S.d. 0.00 0.00
day 7 N 5 5
  Median 0.00 0.00
EO BAL% Mean 0.25 x+ 0.00
[%] S.d. 0.31 0.00
day 7 N 5 5
  Median 0.25 0.00
ATY BAL% Mean 0.00 x+ 0.05
[%] S.d. 0.00 0.11
day 7 N 5 5
  Median 0.00 0.00
TCN BAL Mean 55.01 x+ 54.00
[cn/µL BAL] S.d. 8.80 5.48
day 28 N 5 5
  Median 57.84 56.48
MPH BAL_C Mean 53.41 x+ 52.07
[cn/µL BAL] S.d. 8.38 7.44
day 28 N 5 5
  Median 53.36 56.06
LY BAL_C Mean 0.43 x+ 0.63
[cn/µL BAL] S.d. 0.46 1.02
day 28 N 5 5
  Median 0.24 0.28
PMN BAL_C Mean 1.08 x+ 1.28
[cn/µL BAL] S.d. 1.36 1.84
day 28 N 5 5
  Median 0.44 0.48
MO BAL_C Mean 0.03 x+ 0.00
[cn/µL BAL] S.d. 0.06 0.00
day 28 N 5 5
  Median 0.00 0.00
EO BAL_C Mean 0.05 x+ 0.02
[cn/µL BAL] S.d. 0.07 0.05
day 28 N 5 5
  Median 0.00 0.00
ATY BAL_C Mean 0.00 x+ 0.00
[cn/µL BAL] S.d. 0.00 0.00
day 28 N 5 5
  Median 0.00 0.00
MPH BAL% Mean 97.20 x+ 96.15
[%] S.d. 2.95 5.98
day 28 N 5 5
  Median 98.50 98.75
LY BAL% Mean 0.75 x+ 1.25
[%] S.d. 0.73 2.11
day 28 N 5 5
  Median 0.50 0.50
PMN BAL% Mean 1.90 x+ 2.55
[%] S.d. 2.33 3.81
day 28 N 5 5
  Median 1.00 1.00
MO BAL% Mean 0.05 x+ 0.00
[%] S.d. 0.11 0.00
day 28 N 5 5
  Median 0.00 0.00
EO BAL% Mean 0.10 x+ 0.05
[%] S.d. 0.14 0.11
day 28 N 5 5
  Median 0.00 0.00
ATY BAL% Mean 0.00 x+ 0.00
[%] S.d. 0.00 0.00
day 28 N 5 5
  Median 0.00 0.00

BAL: Cytokines
Air control; males

30 mg/m3
males
MCP-1 BAL Mean 14.0 x+ 14.0
[pg/mL] S.d. 0.0 0.0
day 7 N 5 5
  Median 14.0 14.0
IL-8 BAL Mean 120.8 x+ 103.3
[pg/mL] S.d. 35.5 30.3
day 7 N 5 5
  Median 109.1 111.2
M-CSF BAL Mean 19 x+ 14
[pg/mL] S.d. 11 0
day 7 N 5 5
  Median 14 14
OSP BAL Mean 121.02 x+ 164.45
[pg/mL] S.d. 77.32 121.13
day 7 N 5 5
  Median 109.30 90.53
MCP-1 BAL Mean 14.0 x+ 16.8
[pg/mL] S.d. 0.0 6.2
day 28 N 5 5
  Median 14.0 14.0
IL-8 BAL Mean 72.7 x+ 99.7
[pg/mL] S.d. 36.6 60.6
day 28 N 5 5
  Median 77.1 81.4
M-CSF BAL Mean 17 x+ 14
[pg/mL] S.d. 6 0
day 28 N 5 5
  Median 14 14
OSP BAL Mean 297.68 x+ 388.70
[pg/mL] S.d. 194.53 216.56
day 28 N 5 5
  Median 198.70 318.20

Applicant's summary and conclusion

Executive summary:

Treatment-related alterations were found in the lungs directly after the end of the exposure period. Histopathology revealed the presence of the test substance in the lungs, which correlated with the discoloration noted at gross pathology. The test substance was observed as fine brownish-orange particles contained predominantly in macrophages. These were diffusely distributed in the lumen of alveoli and terminal bronchioles, with a tendency to concentrate in the region of the terminal bronchioles. Furthermore, a minimal hypertrophy/hyperplasia was in general observed in the bronchioles but it was most predominant at the level of the terminal bronchioles and alveolar ducts.

Minimal to slight amounts of free pigment particles were found in the lumen of the larynx and nasal cavity without generating any observable lesions. Minimal number of pigment-laden macrophages appeared also in the mediastinal and tracheobronchial lymph nodes, as part of the clearance function.

At the end of the 3-week recovery period no organ weight alterations were found. The discoloration of the lungs caused by the test substance persisted. Histopathology revealed in the lungs a full regression of the hypertrophy/hyperplasia in bronchioles, terminal bronchioles and alveolar ducts. The number of luminal pigment-laden macrophages was diminished but had not disappeared. At the same time a minimal increase in the number of pigment-laden macrophages in the bronchus-associated lymphoid tissue (BALT) and in the tracheobronchial lymph nodes was observed, suggesting a minimal clearance of the test substance from the lung to the regional lymph nodes. All of these histopathological findings are regarded as treatment-related but since no tissue injury was present in the examined respiratory organs, all of them are considered to be adaptive and not adverse. No findings were observed in the trachea and mediastinal lymph node.