29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper

Endpoint summary

Description of key information

Oral: sub-chronic repeated dose toxicity study in rats (similar to OECD TG 408; pre-GLP): NOAEL = ca. 4500 mg/kg bw/d (highest tested dose)

Inhalation: short-term inhalation lung toxicity study in rats: NOAEC = 30 mg/m³ (highest tested concentration)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken. Extra investigation on copper content in liver and kidneys included.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: The chemical analysis, performed at Midwest Research Institute indicated that the purity was 104.7 % +- 1.1 % (elemental analysis)

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries
- Weight at study initiation: males: 72 - 94 g; females: 70 - 90 g
- Housing: polycarbonate cages: groups of 5 rats per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: feed

Vehicle:

other: 12 % water was added to the test material as a dust control agent

Details on oral exposure:

Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. 12 % water was added to the test material as a dust control agent prior to mixing with the meal. For each dose level, one weekly lot of 4500 g (+ 12 % water compensation) was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 252 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 126 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 63 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 30.24 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 15.12 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.
One analysis was perfomed to determine the accuracy of the mixture concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One analysis was performed to determine the accuracy of mixture concentrations; results were within +/- 10 % of the desired dose concentration.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Dose / conc.:

0.3 other: % in the diet

Dose / conc.:

0.6 other: % in the diet

Dose / conc.:

1.25 other: % in the diet

Dose / conc.:

2.5 other: % in the diet

Dose / conc.:

5 other: % in the diet

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- Five dose levels of 0.0, 0.3, 0.6, 1.25, 2.5 and 5.0 % in feed were used in this study (approx. 0, 250, 500, 1100, 2200 and 4500 mg/kg bw for both sexes [based on 16.4 g/d average food consumption, 0.182 kg average bw for males and on 11.55 g/d average food consumption, 0.130 kg average bw] for females).
- The selected doses were prepared by mixing together weighed portions of Purina Lab Chow in meal form with weighed portions of the chemical. 12 % water was added to the chemical as a dust control agent prior to mixing with the meal.
- Each dosed group received 91 consecutive days of dosed feed mixture.

Observations and examinations performed and frequency:

Animals were observed twice each day for clinical signs, with at least six hours between observations. All clinical signs were recorded daily. Additional studies included blood sampling for the animal disease screening program from 10 control rats, 5 males and 5 females.

Sacrifice and pathology:

- Rats were necropsied on day 92 and 93.
- Gross examination were performed on all animals from all dosage groups.
- Microscopic examinations were performed on all tissues from all animals in the control group and the highest dose treatment group: Kidney, liver, lung, heart, pancreas and pituitary.

Other examinations:

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male rats in the highest dose group (5 % w/w) and control groups. See details and results in endpoint "7.1.1. Basic toxicokinetics" in endpoint study record "Batelle 76-34-106002, rat".

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight data showed differential weight gains in dosed rats no greater than 10 % to the control group; except in the highest dose level females which had + 12 % differential gain. However, no trend was established through the groups in either males or females. The greatest range between any two female groups was 12 % to - 5 % between the two highest doses (5 % and 2 .5 %). The greatest range between any two male groups was 9 % to -9 % between the highest dose and the third highest dose (5 % and 1 .25 %).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically the observed lesions were encountered in both the control and experimental high dose group with similar frequency and severity . The high incidence of pulmonary lesions in both groups is thought to be associated with Sendai virus infection. No histopathologic lesions considered to be compound-related were encountered in the tissues examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse findings

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No accumulation of copper in liver or kidneys

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4 500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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:

guideline study

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)

Limit test:

yes

Specific details on test material used for the study:

Blue powder
stored at room temperature

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; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation (means): Main group day 0: control group: 252 g; test groups: 252 - 254g
- 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.

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: see table 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. Mean relative humidities in the inhalation systems ranged between 39.6 and 53.5 %. Mean
temperatures in the inhalation systems ranged between 21.0 and 22.2°C.

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

Dose / conc.:

29.3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
29.3 +/- 3.4 mg/m³
Basis:
analytical conc.

Dose / conc.:

9.7 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
9.7 +/- 1.7 mg/m3
Basis:
analytical conc.

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
3.0 +/- 0.7 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.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

One animal exposed to the low concentration died on study day 4 after exposure. As no other animals exposed to higher concentrations died, the death was considered not substance-related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological 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):

Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m³) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m³). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m³) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m³) in cecum and rectum.
All discolorations revealed the presence of the pigment and were regarded as treatment-related. In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
One animal showed at necropsy a red discoloration in the skin of the snout.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see table 3

All of these findings were regarded as treatment-related and were considered to be adaptive and not adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Acute phase proteins
Neither after the administration of the compound nor after the recovery period there were any alteration of the serum haptoglobin and alpha-2-macroglobulin levels.

Bronchoalveolar lavage fluid (BAL)
No treatment-related changes among bronchoalveolar cytology parameters were observed.

Total protein and enzymes in BAL
No treatment-related changes among BAL enzyme activities and total protein levels were observed.

Antigens in BAL
No treatment-related changes among BAL antigen levels were observed.

Gross necropsy
Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m3) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m3). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m3) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m3) in cecum and rectum. All discolorations revealed the presence of the pigment and were regarded as treatment-related.
In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
Animal No. 11 (deceased) showed at necropsy a red discoloration in the skin of the snout.
At the end of the recovery period, the blue discoloration of the lung was still visible.

Histopathology (see tables 2-4)
After exposure, in test group 3 and 2 (30 and 10 mg/m3), slight and minimal number of macrophages showing blue pigment-laden cytoplasms (histiocytosis with pigment particles) were seen in the alveoli with a tendency to accumulate in lumen of the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). Minimal epithelial hypertrophy and/or hyperplasia was noted in terminal bronchioles of 2 out of 3 males of test group 3 (30 mg/m3). This finding was characterized by the presence of high cuboidal epithelial cells, accompanied by a slight cytoplasmic basophilia. Single alveolar macrophages containing very few blue cytoplasmic pigment particles were seen in animals of test group 1 (3 mg/m3). A minimal number of pigmentladen macrophages was observed in the bronchus-associated lymphoid tissue (BALT) at all concentration levels (10, 30 and 300 mg/m3) without a dose-dependency.
At the end of the recover period all males of test group 3 (30 mg/m3) and 2 out of 3 males of test group 2 (10 mg/m3) showed a minimal number of pigment-laden alveolar macrophages with a tendency to accumulate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). In one male out of 3 of test group 2 (10 mg/m3) and all males of test group 1 (3 mg/m3) single macrophages containing very few cytoplasmic pigment particles were found in alveolar lumina. The bronchus-associated lymphoid tissue (BALT) of all males in test group 2 and 3 showed minimal number of pigment-laden macrophages. Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3) and in the tracheobronchial lymph node of one out of 3 animals of test group 2 (10 mg/m3).

In the larynx of animals of test group 3 (30 mg/m3), minimal focal epithelial alteration was seen at the base of the epiglottis (level I). In general, minimal amount of pigment particles (admixed with mucus secretion or within macrophages) were observed in the lumen of some animals in the larynx and nasal cavity.

Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3). All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Deceased animal: One animal showed in the lungs single alveolar macrophages containing very few cytoplasmic pigment particles and minimal number of pigment-laden macrophages in the BALT (bronchus-associated lymphoid tissue). These findings were regarded as treatment-related and adaptive and revealed the presence of the test-substance. In the larynx, minimal epithelial alteration (level I) and minimal focal squamous metaplasia (level II) were seen, which were assessed as treatment-related and adaptive (Kaufmann et al., 2009). No histopathological findings were detected in the other examined organs that can explain the death of this animal.

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air

Based on:

test mat.

Remarks:

mean measured concentration

Sex:

male

Basis for effect level:

other: No adverse effects observed at the highest tested dose

Critical effects observed:

no

Particle size distribution was measured by different devices. The data are presented in table 1 below and in the attached pdf file. Cascade impactor measurements showed test atmospheres with high fraction of respirable particles. It seems that MMAD decreased slightly with increasing atmospheric dust concentration. APS measurements in the atmospheres of test groups 1 and 2 showed comparable MMADs to those of cascade impactor. The data of test group 3 showed higher MMAD than in test group 1 and 2, whereas cascade impactor measurements showed a tendency other way round.

The count median diameters measured by WELAS and SMPS showed the fine nature of the dust in the test atmospheres. Particle count concentrations increased with increasing mass concentrations. Slightly higher count median diameters were measured by WELAS, which is in the nature of the different measuring principle of these two devices. In general, the measured values by WELAS and SMPS were consistent. Both showed a slightly lower count median diameter of dust in test group 3 than in the groups 1 and 2, as observed in MMADs measured by cascade impactor. Summarizing the particle size measured by different devices, the test atmospheres consisted of respirable particles. The MMAD of test group 3 was slightly lower than in test groups 1 and 2. With increasing test concentration, higher forces applied during dust generation pressing the test material against the rotating brush might have caused a higher shear force on agglomerates. Thus, smaller particles may be generated. Same tendency was showed by

WELAS and SMPS. APS was the only one device showing higher MMAD in test group 3 than in the others. This phenomena cannot be explained.

Table 1 Particle size distributions measured by different devices

Test groups	1	2	3
Target concentration (mg/m3)	3	10	30
Marple cascade impactor MMAD (µm) / GSD	1.1 µm / 3.6 1.1 µm / 2.8	0.8 µm / 2.6 0.7 µm / 2.7	0.5 / 4.9 0.6 / 2.8
APS 3321 MMAD (µm) / GSD	0.94 µm / 1.851 0.92 µm / 1.591 0.91 µm / 1.501 0.91 µm / 1.652 0.95 µm / 2.562 0.91 µm / 1.642	1.01 µm / 3.161 0.94 µm / 1.911 0.96 µm / 2.351 1.04 µm / 3.522 0.98 µm / 2.332 0.98 µm / 2.202	2.46 µm / 2.421 2.59 µm / 2.501 2.64 µm / 2.541 2.41 µm / 2.422 2.89 µm / 2.812 2.62 µm / 2.452
WELAS 2000 (µm): Count median diameter (Q0)	0.404	0.407	0.379
SMPS (µm): Count median diameter (Q0)	0.281	0.315	0.257
Count concentration of particles in WELAS 2000 (number particle/cm3)	7327	16555	171154
Count concentration of particles in SMPS (number particle/cm3)	18909	76637	454053

1 = measured on 25 Feb 2014

2 = measured on 26 Jun 2014

Table 2: Macroscopic findings

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Cecum
·   Discoloration of contents			2	3
Glandular stomach
·   Discoloration of contents		1	2	2
Jejunum
·   Discoloration of contents		1	1	3
Lungs
·   Discoloration			3	3
Rectum
·   Discoloration of contents			1	1
Skin
·   Discoloration		2	3	3

Table 3: Microscopic findings at the end of exposure

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden			3	3
·   Grade 1			3
·   Grade 2				3
Pigment particles, in single macrophages		3
·   Present		3
Hypertrophy/hyperplasia, terminal bronchiole				2
·   Grade 1				2
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden		3	2	1
·   Grade 1		3	2	1

Table 4: Microscopic findings at the end of recovery

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden	0	0	2	3
·   Grade 1			2	3
Pigment particles, in single macrophages		3	1
·   Present		3	1
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden	0	0	3	3
·   Grade 1			3	3

Conclusions:

Inhalation exposure of rats to up 30 mg/m³ test substance on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract. Some non-adverse adaptive changes fully regressed within a 3-week
recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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:

guideline study

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)

Limit test:

yes

Specific details on test material used for the study:

Blue powder
stored at room temperature

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; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation (means): Main group day 0: control group: 252 g; test groups: 252 - 254g
- 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.

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: see table 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. Mean relative humidities in the inhalation systems ranged between 39.6 and 53.5 %. Mean
temperatures in the inhalation systems ranged between 21.0 and 22.2°C.

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

Dose / conc.:

29.3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
29.3 +/- 3.4 mg/m³
Basis:
analytical conc.

Dose / conc.:

9.7 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
9.7 +/- 1.7 mg/m3
Basis:
analytical conc.

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
3.0 +/- 0.7 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.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

One animal exposed to the low concentration died on study day 4 after exposure. As no other animals exposed to higher concentrations died, the death was considered not substance-related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological 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):

Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m³) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m³). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m³) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m³) in cecum and rectum.
All discolorations revealed the presence of the pigment and were regarded as treatment-related. In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
One animal showed at necropsy a red discoloration in the skin of the snout.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see table 3

All of these findings were regarded as treatment-related and were considered to be adaptive and not adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Acute phase proteins
Neither after the administration of the compound nor after the recovery period there were any alteration of the serum haptoglobin and alpha-2-macroglobulin levels.

Bronchoalveolar lavage fluid (BAL)
No treatment-related changes among bronchoalveolar cytology parameters were observed.

Total protein and enzymes in BAL
No treatment-related changes among BAL enzyme activities and total protein levels were observed.

Antigens in BAL
No treatment-related changes among BAL antigen levels were observed.

Gross necropsy
Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m3) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m3). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m3) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m3) in cecum and rectum. All discolorations revealed the presence of the pigment and were regarded as treatment-related.
In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
Animal No. 11 (deceased) showed at necropsy a red discoloration in the skin of the snout.
At the end of the recovery period, the blue discoloration of the lung was still visible.

Histopathology (see tables 2-4)
After exposure, in test group 3 and 2 (30 and 10 mg/m3), slight and minimal number of macrophages showing blue pigment-laden cytoplasms (histiocytosis with pigment particles) were seen in the alveoli with a tendency to accumulate in lumen of the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). Minimal epithelial hypertrophy and/or hyperplasia was noted in terminal bronchioles of 2 out of 3 males of test group 3 (30 mg/m3). This finding was characterized by the presence of high cuboidal epithelial cells, accompanied by a slight cytoplasmic basophilia. Single alveolar macrophages containing very few blue cytoplasmic pigment particles were seen in animals of test group 1 (3 mg/m3). A minimal number of pigmentladen macrophages was observed in the bronchus-associated lymphoid tissue (BALT) at all concentration levels (10, 30 and 300 mg/m3) without a dose-dependency.
At the end of the recover period all males of test group 3 (30 mg/m3) and 2 out of 3 males of test group 2 (10 mg/m3) showed a minimal number of pigment-laden alveolar macrophages with a tendency to accumulate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). In one male out of 3 of test group 2 (10 mg/m3) and all males of test group 1 (3 mg/m3) single macrophages containing very few cytoplasmic pigment particles were found in alveolar lumina. The bronchus-associated lymphoid tissue (BALT) of all males in test group 2 and 3 showed minimal number of pigment-laden macrophages. Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3) and in the tracheobronchial lymph node of one out of 3 animals of test group 2 (10 mg/m3).

In the larynx of animals of test group 3 (30 mg/m3), minimal focal epithelial alteration was seen at the base of the epiglottis (level I). In general, minimal amount of pigment particles (admixed with mucus secretion or within macrophages) were observed in the lumen of some animals in the larynx and nasal cavity.

Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3). All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Deceased animal: One animal showed in the lungs single alveolar macrophages containing very few cytoplasmic pigment particles and minimal number of pigment-laden macrophages in the BALT (bronchus-associated lymphoid tissue). These findings were regarded as treatment-related and adaptive and revealed the presence of the test-substance. In the larynx, minimal epithelial alteration (level I) and minimal focal squamous metaplasia (level II) were seen, which were assessed as treatment-related and adaptive (Kaufmann et al., 2009). No histopathological findings were detected in the other examined organs that can explain the death of this animal.

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air

Based on:

test mat.

Remarks:

mean measured concentration

Sex:

male

Basis for effect level:

other: No adverse effects observed at the highest tested dose

Critical effects observed:

no

Particle size distribution was measured by different devices. The data are presented in table 1 below and in the attached pdf file. Cascade impactor measurements showed test atmospheres with high fraction of respirable particles. It seems that MMAD decreased slightly with increasing atmospheric dust concentration. APS measurements in the atmospheres of test groups 1 and 2 showed comparable MMADs to those of cascade impactor. The data of test group 3 showed higher MMAD than in test group 1 and 2, whereas cascade impactor measurements showed a tendency other way round.

The count median diameters measured by WELAS and SMPS showed the fine nature of the dust in the test atmospheres. Particle count concentrations increased with increasing mass concentrations. Slightly higher count median diameters were measured by WELAS, which is in the nature of the different measuring principle of these two devices. In general, the measured values by WELAS and SMPS were consistent. Both showed a slightly lower count median diameter of dust in test group 3 than in the groups 1 and 2, as observed in MMADs measured by cascade impactor. Summarizing the particle size measured by different devices, the test atmospheres consisted of respirable particles. The MMAD of test group 3 was slightly lower than in test groups 1 and 2. With increasing test concentration, higher forces applied during dust generation pressing the test material against the rotating brush might have caused a higher shear force on agglomerates. Thus, smaller particles may be generated. Same tendency was showed by

WELAS and SMPS. APS was the only one device showing higher MMAD in test group 3 than in the others. This phenomena cannot be explained.

Table 1 Particle size distributions measured by different devices

Test groups	1	2	3
Target concentration (mg/m3)	3	10	30
Marple cascade impactor MMAD (µm) / GSD	1.1 µm / 3.6 1.1 µm / 2.8	0.8 µm / 2.6 0.7 µm / 2.7	0.5 / 4.9 0.6 / 2.8
APS 3321 MMAD (µm) / GSD	0.94 µm / 1.851 0.92 µm / 1.591 0.91 µm / 1.501 0.91 µm / 1.652 0.95 µm / 2.562 0.91 µm / 1.642	1.01 µm / 3.161 0.94 µm / 1.911 0.96 µm / 2.351 1.04 µm / 3.522 0.98 µm / 2.332 0.98 µm / 2.202	2.46 µm / 2.421 2.59 µm / 2.501 2.64 µm / 2.541 2.41 µm / 2.422 2.89 µm / 2.812 2.62 µm / 2.452
WELAS 2000 (µm): Count median diameter (Q0)	0.404	0.407	0.379
SMPS (µm): Count median diameter (Q0)	0.281	0.315	0.257
Count concentration of particles in WELAS 2000 (number particle/cm3)	7327	16555	171154
Count concentration of particles in SMPS (number particle/cm3)	18909	76637	454053

1 = measured on 25 Feb 2014

2 = measured on 26 Jun 2014

Table 2: Macroscopic findings

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Cecum
·   Discoloration of contents			2	3
Glandular stomach
·   Discoloration of contents		1	2	2
Jejunum
·   Discoloration of contents		1	1	3
Lungs
·   Discoloration			3	3
Rectum
·   Discoloration of contents			1	1
Skin
·   Discoloration		2	3	3

Table 3: Microscopic findings at the end of exposure

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden			3	3
·   Grade 1			3
·   Grade 2				3
Pigment particles, in single macrophages		3
·   Present		3
Hypertrophy/hyperplasia, terminal bronchiole				2
·   Grade 1				2
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden		3	2	1
·   Grade 1		3	2	1

Table 4: Microscopic findings at the end of recovery

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden	0	0	2	3
·   Grade 1			2	3
Pigment particles, in single macrophages		3	1
·   Present		3	1
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden	0	0	3	3
·   Grade 1			3	3

Conclusions:

Inhalation exposure of rats to up 30 mg/m³ test substance on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract. Some non-adverse adaptive changes fully regressed within a 3-week
recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

In two 90 day subchronic studies with F344 rats and B6C3F1 mice, comparable to OECD guideline 408, male and female animals received dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) copper phthalocyanine, administered in the food (approx. 0, 250, 500, 1100, 2200 and 4500 mg/kg bw for rats of both sexes, approx. 0, 1000, 2000, 4000, 8000 and 16000 mg/kg bw for male mice, and approx. 0, 1100, 2200, 4700, 9400 and 18700 mg/kg bw for female mice, calculated on average food consumption for 91 consecutive days) (Batelle 1979). Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All clinical signs were recorded daily. Gross examinations were performed on all animals from all dosage groups. Microscopic examinations were performed from kidney, liver, lung, heart, pancreas and pituitary from all animals in the control group and the highest dose treatment group. No clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken. No substance related mortality was reported for rats.There were seven early deaths for mice. Four male mice from four different dose or control groups and 3 control female mice died during the study. During the course of the 91-day study, neither treatment related signs of toxicity, nor abnormal clinical signs were observed in both rats and mice. Diet consumption among both male and female rats did not vary widely, nor showed any dose related trends. In mice, there were also no trends in diet consumption among dosed animals compared with controls in either male or female mice. No substance related changes were reported on macroscopic and histopathological examination of both rats and mice.

Therefore, the NOAEL was determined at the highest tested doses of approx. 4500 mg/kg bw/d in rats and ca. 16000 mg/kg bw/d in mice.

A 28 day subacute study was conducted, according to the Japanese guideline for 28 Day Repeated Dose Toxicity Test of Chemicals with Wistar rats, which received 0, 40, 200 and 1000 mg/kg/day test substance by gavage (JETOC 1997). Food consumption and body weight were determined. The animal’s state of health was checked by cage side observations as well as by detailled clinical observations. Clinicochemical and hematological examinations were carried out at the end of the administration period and after a recovery period of 14 days. All animals were subjected to gross-pathological assessment, followed by histopathological examination. No changes in general condition, body weight gain or food consumption were detected in any of the groups. After the 28 days of administration, a slight, but significant decrease in red blood cell count (RBC) and decrease of hemoglobin (Hb) and packed cell volume (PCV) were detected in the 200 and 1000 mg/kg male groups. These slight changes were dose dependent. After the recovery period, a significant increase of erythroblasts was detected in the 1000 mg/kg female group. Increases of absolute organ weights of lung, spleen, adrenal and salivary gland and a tendency for increased relative organ weights of the spleen were evident in the 1000 mg/kg male group. No histopathological changes due to administration of the test substance were detected. Therefore, a NOEL of 40 mg/kg bw per day was determined for male and female rats under the test conditions chosen. However, given the minor severity of the effects, their reversibility and the restricted relevance of the observations, a NOAEL of 1000 mg/kg bw may be attributed.

Inhalation:

The (mono)chlorinated form of the test substance (CAS 12239-87-1) was tested in a short-term inhalation study in rats (BASF SE 2016). This GLP-compliant non-guideline study was designed to investigate local and systemic effects both after a five-day exposure and after a treatment free recovery period of 28 days. The substance was tested at concentrations of 3, 10 and 30 mg/m³ as all available data indicates that it behaves like an inert organic dust. Concerning clinical pathology no treatment-related, adverse effects were observed up to a concentration of 30 mg/m³.

Presence of the blue pigment was visible in the lungs at the mid and the high dose group and in the content of stomach and intestine at all dose groups. In the lung, blue staining correlated with pigment-laden macrophages.

Regarding pathology, treatment-related findings were observed in the lungs, larynx and lymph nodes of the main group. In the lungs, a concentration-dependent increase in the amount of macrophages and the phagocytized blue pigment was seen in all animals starting from 3 mg/m³. This finding, which was mainly confined to the lumen of the bronchiole-alveolar junction lumina, reached only a slight grading at 30 mg/m³. It was considered to be part of the clearance function of the test substance and was therefore regarded as non-adverse. A minimal epithelial hypertrophy and/or hyperplasia were observed in the terminal bronchioles of 2 out of 3 animals at 30 mg/m³, without signs of cell death or inflammation. Based on the low magnitude of this finding affecting only 2 out of 3 animals and the lack of treatment-related findings in the bronchoalveolar lavage fluid, it was considered an adaptive response. Minimal pigment-laden macrophages observed either in the BALT (bronchus-associated lymphoid tissue) at all concentrations levels as well as very few pigment particles within single macrophages seen in the tracheobronchial and mediastinal lymph nodes at 30 mg/m³ were also regarded as part of the clearance function and were therefore assessed as non-adverse.

In the larynx of animals of test group 3 (30 mg/m³), minimal focal epithelial alteration, characterized by loss of cilia and flattening of the epithelial surface, was seen in the ventral pouch of the epiglottis. This finding was considered to be treatment-related and adaptive, but can be also found as a background lesion (Kaufmann et al., 2010).

At the end of the 3-week recovery period, the epithelial hypertrophy and/or hyperplasia in the terminal bronchioles at 30 mg/m³ had completely regressed, implying the adaptive nature of this change. Furthermore, the number of pigment-laden macrophages showed a tendency to decrease in test groups 2 and 3 (10 and 30 mg/m³) when compared with the main group. At the same time the number of pigment-laden macrophages in the bronchus-associated lymphoid tissue (BALT) as well as the single macrophages with very few pigment particles in the mediastinal and tracheobronchial lymph nodes revealed normal features of continuing clearance of the test substance. All of these findings were regarded as treatment-related and were considered to be adaptive and not adverse. Thus, a NOAEC of 30 mg/m³ (highest tested concentration) was determined.

Kaufmann W., Bader R., Ernst H., Harada T., Hardisty J., Kittel B., Kolling A., Pino M., Renne R., Rittinghausen S., Schulte A., Wöhrman T. and Rosenbruch M. (2009) 1st International ESTP-Workshop: “Larynx squamous metaplasia”. A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment. Exp Toxicol Pathol 61:591 - 603

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.