3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Aug 2020 - 23 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

25 June 2018

Deviations:

yes

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:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

EC Number:

250-063-5

EC Name:

3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

Cas Number:

30125-47-4

Molecular formula:

C26H6Cl8N2O4

IUPAC Name:

4,5,6,7-tetrachloro-2-[2-(4,5,6,7-tetrachloro-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)quinolin-8-yl]-1H-isoindole-1,3(2H)-dione

Test material form:

solid: nanoform

Details on test material:

- Physical state/ appearance: solid / yellow
- Shape of particles: spherical
- Aspect ratio: 1.5
- Particle size distribution: 62.7 nm
- Crystal structure: crystalline
- Surface area of particles: 29.6 m^2/g
- Surface treatment: no

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

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):
- Housing: The rats were housed together (up to 5 animals per cage)
- Diet: Mouse/rat laboratory diet; ad libitum
- 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

Mass median aerodynamic diameter (MMAD):

>= 0.42 - <= 0.67 µm

Remarks on MMAD:

Please, see Table 1 in section "Any other information on materials and methods incl. tables"

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) was 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.

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please, see Table 2 in section "Any other information on materials and methods incl. tables"

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, for five consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/m³ air

Remarks:

Measured concentration: 5.1 +/- 0.2 mg/m³

Doses / concentrations 2

Dose / conc.:

20 mg/m³ air

Remarks:

Measured concentration: 19.3 +/- 0.4 mg/m³

Doses / concentrations 3

Dose / conc.:

60 mg/m³ air

Remarks:

Measured concentration: 60.6 +/- 2.0 mg/m³

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Examinations

Observations and examinations performed and frequency:

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

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

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

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

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

CLINICAL CHEMISTRY: 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 . Rat haptoglobin was measured with an ELISA.

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. 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 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. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA , Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA, Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA , Rodent osteopontin measured with an ELISA .

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: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means.
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
- Broncho-alveolar lavage fluid (BALF): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period and the post-exposure observation period the animals showed no clinical signs and findings different from normal, as well as the animals of the control group and low concentration (5 mg/m³) during the exposure period. At mid (20 mg/m³) and high (60 mg/m³) the animals showed substance contaminated fur after daily exposure.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically
significantly different from the control group 0.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No substance-related changes of food consumption were observed during the whole study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
At the end of the administration period, in males of test group 3 (60 mg/m3) absolute and relative neutrophil counts were significantly increased. However, the values were marginally above the historical control range (absolute neutrophils) or within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 10.0-17.9 %). Therefore, these alterations were regarded as incidental and not treatment related

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
All mean absolute weight parameters did not show significant differences when compared to the control group.
When compared to control group (=100%), the mean relative weights of the adrenals were significantly increased in group 2 (20 mg/m³): 118 %; but not in the other dose groups. Due to a missing dose-response relationship the increase was regarded as incidental.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Recovery groups:
All mean absolute weight parameters did not show significant differences when compared to the control group.
Due to a missing dose-response relationship the decrease in relative thymus weight (73 %, 20 mg/m³) was regarded as incidental. The increased relative thyroid glands weight (126 %, 60 mg/m³) was within historical control values and therefore as well considered as not treatment-related.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All animals of test group 3 (60 mg/m³) of the main and recovery group revealed a yellow discoloration in the lungs, mediastinal and tracheobronchial lymph nodes. This was regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Description (incidence and severity):

Main group:
In the lungs animals of test group 2 and 3 (20 and 60 mg/m³) showed increased numbers of histiocytes (histiocytosis) within the alveoli which revealed yellow particles within their cytoplasm. In test group 1 (5 mg/m³) the histiocytes were not increased in number but also contained yellow particles inside.
All animals of test group 3 (60 mg/m³) had in addition to the increased numbers of histiocytes also minimal infiltration with neutrophils and a hypertrophy/hyperplasia of the epithelium of the terminal bronchioles.
In some animals of all treated groups occasionally single macrophages with yellow particles in their cytoplasm were observed intravascularly. All these findings were regarded to be treatment-related.
At the base of the epiglottis of all animals of test group 3 (60 mg/m³) a minimal focal area with flattened epithelium and loss of cilia was seen in three of the five animals. This finding was regarded to be treatment related.
The tracheobronchial lymph node was chosen exemplarily, similar findings were observed in the mediastinal lymph nodes. Within the lymph node of animals of all test groups histiocytes were observed containing the same yellow particles inside their cytoplasm as observed in the lungs. This was regarded to be treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Recovery group:
In the lung animals of test group 3 (60 mg/m³) showed increased numbers of histiocytes (histiocytosis) which revealed yellow particles within their cytoplasm. In test group 1 and 2 (5 and 20 mg/m³) the histiocytes were not increased in number but also contained yellow particles inside. Some animals of all treated groups showed single macrophages with yellow particles in
the bronchus-associated lymphoid tissue (BALT).
In some animals of test groups 2 and 3 (20 and 60 mg/m³) occasionally single macrophages with yellow particles in their cytoplasm were observed intravascularly. All these findings were regarded to be treatment-related.
Some animals of all treated groups still had macrophages with yellow particles within the draining lymph nodes of the lung. In test group 3 (60 mg/m³) these macrophages had formed small aggregates in addition. These findings were regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL):
At the end of the administration period, in males of test group 3 (60 mg/m3) total cell counts in BAL were significantly increased. This increase was mainly due to increased absolute neutrophil counts followed by increases of absolute lymphocyte and monocyte counts in BAL (not statistically significant). Additionally, in the BAL of these individuals, higher levels of bronchial epithelial cells were found (not statistically significant). These changes were regarded as treatment related and adverse.
After three weeks of recovery, in male rats of test group 1 (5 mg/m3) absolute lymphocyte counts in BAL were significantly increased, and in males of test group 2 (20 mg/m3) relative neutrophil counts in BAL were significantly higher compared to study controls. Additionally, in males of test groups 2 and 3 (20 and 60 mg/m3) relative macrophage counts were significantly decreased. However, all mentioned changes in this paragraph were not dose dependent and therefore, they were regarded as incidental and not treatment related.
At the end of the administration period, in males of test group 3 (60 mg/m3) total protein levels in BAL γ-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities in BAL were significantly increased. Alkaline phosphatase (ALP) and N-Acetyl-β-D-glucosaminidase (NAG) activities in BAL were also significantly increased in this test group, but ALP was only marginal higher compared to controls, and the NAG increase was below 2fold. Therefore, total protein level as well as GGT, LDH and ALP activity increases in BAL of test group 3 were regarded as treatment-related and adverse, whereas the NAG activity increase was regarded as maybe treatment-related but non-adverse.
After the 3-week recovery period, no significant changes of the mentioned parameters occurred.

Antigens in BAL:
After the administration period, in males of test group 3 (60 mg/m3) monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL were significantly increased. These alterations were regarded as treatment related and adverse.
After the recovery period, in males of test group 2 (20 mg/m3) CINC-1/IL-8 levels in BAL were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

>= 60 mg/m³ air

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

20 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Please see section "Any other information on results incl. tables"

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The following local effects were observed after treatment with 60 mg/m^3:

• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial epithelial cells in BAL


• Increased total protein levels as well as γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL


• Increased monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL


• Increased histiocytes with intracytoplasmic yellow particles,


• hypertrophy/ hyperplasia of terminal bronchioles,


• infiltration of neutrophils

Data from lavage an histology were consistent with the above mentioned effects. All effects were reversible after a 3-week recovery period.

 

Table 4: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
Total Cells	1.2	1.4	3.7**	1.0	0.8	0.8
Eosinophils	0.1	1.5	0.2	+	+	+
Lymphocytes	1.9	1.8	7.5	7.1*	5.7	2.8
Macrophages	1.2	1.3	2.7	1.0	0.8	0.8
Neutrophils	0.7	2.6	79.0	1.0	3.3	1.8
Monocytes	0.0	3.4	121.6	1.0	1.0	1.8
Epithelial cells	2.1	29.2	50.3	+	+	+

One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

+ increase could not be calculated because of zero activity in controls

 

Table 5: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure)

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
Total Protein	1.2	1.3	2.3**	1.3	1.2	1.2
GGT	1.3	1.4	3.7**	1.0	0.9	0.9
LDH	1.7	1.8	5.0**	1.6	1.2	1.3
ALP	1.1	1.0	2.3**	1.1	1.1	1.0
NAG	1.0	1.4	1.8*	1.3	1.0	1.2

GGT = gamma-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = N-Acetyl-beta-D-glucosaminidase, One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

 

Table 6: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
MCP-1	1.3	1.0	7.5**	0.8	1.0	1.2
CINC-1/IL-8	1.0	0.8	2.3**	0.8	1.6**	1.2
M-CSF	1.0	1.0	1.3	1.0	1.0	1.0
Osteopontin	0.3	0.7	2.4	0.3	1.2	1.2

BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1; MCP-1 = monocyte chemoattractant protein-1; M-CSF = macrophage colony stimulating factor

One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

 

Table 7: Relative changes of relative organ weights in main group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Adrenal glands	98%	118%*	117%

*p ≤ 0.05; **p ≤ 0.01

 

Table 8: Relative changes of relative organ weights in recovery group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Thymus	82%	73%*	90%
Thyroid glands	111%	96%	126%*

*p ≤ 0.05; **p ≤ 0.01

 

Table 9: Gross lesions observed in main group animals (F1)

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs ·        Discoloration	0	0	5	5
Mediastinal lymph node ·        Discoloration	0	0	0	5
Tracheobronchial lymph node ·        Discoloration	0	0	0	5

 

Table 10: Gross lesions observed in recovery group animals (R1)

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs ·        Discoloration	0	0	0	5
Mediastinal lymph node ·        Discoloration	0	0	0	5
Tracheobronchial lymph node ·        Discoloration	0	0	0	5

 

Table 11: Incidence and severity of histological findings in the lung of main group animals (F1)

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Histiocytosis, alveolar with particles	0	0	5	5
·        Grade 1			5
·        Grade 2				5
Particles within histiocytes	0	5	0	0
Infiltration neutrophils (m)f	0	0	0	5
·        Grade 1				5
Hypertrophy/hyperplasia term. bronchi	0	0	0	5
·        Grade 1				3
·        Grade 2				2
Macrophages w. particles iv	0	2	4	3

All findings that were not graded were recorded as present

 

Table 12: Incidence and severity of histological findings in the larynx of main group animals (F1)

Larynx (level I)	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Epithelial alteration, (multi)focal	0	0	0	3
·        Grade 1				3

 

Table 13: Incidence and severity of histological findings in the tracheobronchial lymph nodes of main group animals (F1)

Tracheobronchial lymph node	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	4	5	5
Macrophages with particles	0	1	3	5
·        Grade 1		1	3	2
·        Grade 2				3

 

Table 14: Incidence and severity of histological findings in the lungs of recovery group animals (R1)

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Histiocytosis, alveolar w. particles	0	0	0	5
·        Grade 2				5
Particles within histiocytes	0	5	5	0
BALT: macrophages with particles	0	3	5	5
·        Grade 1		3	5	5
Macrophages w. particles iv	0	0	1	3

All findings that were not graded were recorded as present

 

Table 15: Incidence and severity of histological findings in the tracheobronchial lymph nodes of recovery group animals (R1)

Tracheobronchial lymph node	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles	0	3	5	1
·        Grade 1		3	4	1
·        Grade 2			1
Macrophage aggregates with particles	0	0	0	4
·        Grade 1				1
·        Grade 2				3

Applicant's summary and conclusion

Conclusions:

Inhalation exposure of rats to 60 mg/m³ of the test substance on 5 consecutive days caused increased lymphocytes, neutrophils, monocyte counts and epithelial cells in bronchoalveolar lavage. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as increased histiocytes with intracytoplasmic yellow particles, hypertrophy/hyperplasia of terminal bronchioles. All effects were reversible within 3 weeks exposure-free recovery period.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 20 mg/m³ for increased histiocytes with intracytoplasmic yellow particles, hypertrophy/ hyperplasia of terminal bronchioles. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ is the lung.

Executive summary:

The purpose of this study was to determine the pulmonary toxicity in rats using a short-term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of the test subatance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.
For this purpose groups of male Wistar rats (10 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 5, 20, and 60 mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

The particle size resulted in MMADs between 0.42 and 0.67 μm with GSDs between 2.50 and 3.59. The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 90 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

 

The following substance-related adverse findings were observed:

 

Test group 3 (60 mg/m³)
• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial
epithelial cells in BAL
• Increased total protein levels as well as γ-glutamyl transferase (GGT), lactate
dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL
• Increased monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced
neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL
• Increased histiocytes with intracytoplasmic yellow particles, hypertrophy/ hyperplasia of
terminal bronchioles, and infiltration of neutrophils

 

Test group 2 (20 mg/m³) and Test group 1 (5 mg/m³)
No adverse effects were observed.

 

Recovery group
No adverse effects were observed regarding pathology findings.