Reaction mass of 3,6-Bis(3-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, 3-(3-Chlorophenyl)-6-(4-chlorophenyl)-2,5-dihydro- pyrrolo[3,4-c]pyrrole-1,4-dione and 3,6-Bis(4-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

Repeated dose toxicity: inhalation

Administrative data

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other: GLP compliant non-guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

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. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Homogeneity: Homogeneous
- Date of production: date of filling 22 Jan 2012
- Physical state/ Appearance: Solid / orange
- Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 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

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 between 0.6 and 0.7 μm with GSDs between 2.6 and 2.8. The calculated mass fraction of particles below MMAD 3 μm was higher than 93.4 %. Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. The remaining fractions may have reached the upper respiratory tract and been deposited there. In general the generated aerosols of the test substance were within the respirable range. APS 3321 showed MMADs which is higher than the measured value by cascade impactor. This difference may be attributed to bouncing-off effect of the particles within the cascade impactor.

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) will be used for generating the dust. The concentration will be 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.

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:

from Monday to Friday to reach 5 exposures.

Doses / concentrations

Remarks:

Doses / Concentrations:
30 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

8/dose/group (main group or recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- On study day 7 after exposure and on study day 25, 3 animals per group and time point were sacrificed.

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

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. After the inhalation period, in rats of test group 1 (12/0047-1, 30 mg/m³) hemoglobin values were lower compared to controls. The hemoglobin mean was marginally below the historical control range (hemoglobin: 8.6-9.7 mmol/L) and marginally (3.4 %) below the study control value. This was the only changed red blood cell parameter in this group. Therefore, this alteration was regarded as incidental and not treatment-related. After the recovery period, no treatment-related changes among hematology parameters were observed.
CLINICAL CHEMISTRY: No treatment-related, adverse changes among clinical chemistry parameters were observed. At study day 7, in rats of test group 1 (12/0047-1, 30 mg/m³), total protein values were lower compared to controls, which was due to lower globulin values. However, compared to historical controls the study control values were also below the ranges (total protein: 59.09-65.07 g/L, globulins: 21.98-28.70 g/L). These were the only changed blood parameters in these rats. Therefore, this alteration was regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). After the administration period, additional significant changes of clinical chemistry parameters were observed in test group 1 (12/0047-1, 30 mg/m³): decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities; decreased albumin levels; decreased calcium levels. However, all these altered values were within historical control ranges (ALT: 0.53-1.08 μkat/L; AST: 1.46-2.51 μkat/L; albumin: 35.28-38.41 g/L; calcium: 2.42-2.70 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related. After the recovery period, no treatment-related 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):
- Cytology in BAL: Not treatment-related, adverse changes among cytological BAL parameters were observed. At study day 7 in rats of test group 1 (12/0047-1, 30 mg/m³), absolute and relative polymorphonuclear cell (i.e., neutrophils) counts in BAL were marginally increased.
- Total Protein and enzymes in BAL: No treatment-related changes of total protein levels as well as enzyme activities in BAL were observed. After the administration period in rats of test group 1 (12/0047-1, 30 mg/m³), γ-glutamyl transferase (GGT) and N-acetyl-β-Glucosaminidase (NAG) activities were higher compared to controls, but the means were within (for GGT) or only marginal above (for NAG) the historical control ranges (GGT: 0-39 nkat/L, NAG: 7-45 nkat/L,). Therefore, these alterations were regarded as incidental and not treatment-related.
- Antigens in BAL: No treatment-related changes among antigen levels in BAL were observed.
PATHOLOGY
-Main group (F1): No statistical evaluation was performed, since only 3 animals per group were tested. When compared with the control group 0, the terminal body weight was minimally decreased (-6%) and the absolute (+7 %) and relative (+14%) adrenal weight was minimally increased. However, these deviations, as well as all other absolute and relative weight parameters are considered to be within the normal range. A slight orange red discoloration was observed in the lungs of all treated animals of test group 1 and in the fur of one of them. The discoloration of the fur is regarded as not adverse; the discoloration of the lungs was associated with histopathological changes. Treatment-related findings were noted in the lungs. The test substance was mainly visualized in the cytoplasm of macrophages as orange fine particles. A slight 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 amount of pigment in the macrophages was also slight. Slight epithelial hypertrophy and/or hyperplasia was primarily found at the level of the terminal bronchioles and alveolar ducts. This finding was characterized by an increased size and number of bronchiolar epithelial cells, accompanied by a slight cytoplasmic basophilia. Similar findings were seen also in some bronchioles, which were more proximal located. In addition, in alveolar ducts, the septa were slightly thickened. In 1 of 3 males of the exposed group, minimal multifocal inflammatory infiltrates were associated with the presence of hemoglobin crystals. This type of lesion is known to occur incidentally in control animals and therefore, it is not considered to be treatment-related. Minimal focal changes in the larynx, such as epithelial alteration or lymphoid infiltrations are regarded to belong to the spectrum of background lesions and are not considered to be treatment-related. No other treatment-related findings were observed in the nasal cavity and larynx with exception of minimal amounts of free pigment particles admixed with normal luminal mucus secretion. 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. All other findings occurred either individually or were biologically equally distributed over the control and treatment group. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Recovery groups (R1): When compared with the control group 0, the absolute lungs weight was increased (+13%) in the exposed test group (test group 1). However, no histopathological correlate was found that can explain this weight increase and therefore it is regarded as incidental and not related to treatment. Compared with the control group 0, a decrease of -25% was noted in the mean relative thymus weight (0.109%). Compared with the historical controls, this weight was slightly below the minimal mean value (0.121%). Since no thymus weight alteration was noted in the main group during the exposure time (F1) and no casual relation can explain this weight deviation after a 28 day recovery period, the decrease of the relative thymus weight is regarded as incidental and not treatment-related. All other absolute and relative weight parameters in exposed males showed no relevant differences when compared with the control group 0 and are considered to be within the normal range. A skin lesion was found in the neck of one treated male. This finding was regarded as incidental and not related to treatment. No other macroscopic changes were detected in the exposed animals. Compared with the control group 0, all males of the exposed group showed minimal number of orange pigment-laden macrophages in the lumen of alveoli and terminal bronchioles with a tendency to accumulate in the lumen of the bronchiole-alveolar junction. The bronchus-associated lymphoid tissue (BALT) of all males also showed minimal number of pigment-laden macrophages. No treatment-related findings were observed in the nasal cavity, larynx and trachea. Minimal and slight number of pigment-laden macrophages was found in the mediastinal (1 of 3 animals) and tracheobronchial (3 of 3 animals) lymph nodes.

Effect levels

Dose descriptor:

conc. level:

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reverslible marginally increased polymorphonuclear neutrophils in bronchea-alveolar lavage fluid; reversible minimal to slight epithelial hypertrophy/hyperplasia in the bronchioles, terminal bronchioles and alveolar ducts

Target system / organ toxicity

Critical effects observed:

not specified