N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

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:

Feb 8 2022 - August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Principles of method if other than guideline:

5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

252-772-5

EC Name:

N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

35869-64-8

Molecular formula:

C40H23Cl3N8O8

IUPAC Name:

N,N'-(2-chloro-1,4-phenylene)bis{4-[(4-chloro-2-nitrophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Batch identification: 0004501436
Purity: 99.4 %
Date of production: 22 Oct 2010
Expiry date: 22 Oct 2022
Storage conditions: room temperature
Physical state/ appearance: solid brown
Water solubility: insoluble
Mass-specific surface area (BET): 44.3 m2/g
/

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks (when supplied)
- Weight at study initiation (means):ca 255g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) 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 Switze rland), ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%) 45 - 65
- Air changes (per hr): 15
Photoperiod (hrs dark / hrs light):: 12h/12h

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.73 - <= 1.05 µm

Geometric standard deviation (GSD):

3

Remarks on MMAD:

The particle size resulted in MMADs between 0.51 and 0.77 µm with GSDs between 2.9 and 3.92 (Table 1). The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 88 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Details on inhalation exposure:

For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system

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 con
centration 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low 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 these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.

The particle size analysis was carried out with a cascade impactor with the following equipment:
• 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 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/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.

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. During the exposure period, one measurement per concentration with 10 repeats each were performed.

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations.

Duration of treatment / exposure:

6h for 5 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5.1 mg/m³ air (analytical)

Remarks:

63853 particles per cm3 (measured by SMPS)

Doses / concentrations 2

Dose / conc.:

20.1 mg/m³ air (analytical)

Remarks:

116362 particles per cm3 (measured by SMPS)

Doses / concentrations 3

Dose / conc.:

59.7 mg/m³ air (analytical)

Remarks:

355121 particles per cm3 (measured by SMPS)

No. of animals per sex per dose:

10 (five for sacrifice after exposure and 5 for sacrifice after recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Results of short-term inhalation studies with other inert organic pigments
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical bioche Particlesmistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

A check for moribund or dead animals was carried out twice per day on working days. A check for moribund or dead animals was carried out once per day on weekends and holidays.

The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.During exposure only a group wise examination was possible.

The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.

Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible

Sacrifice and pathology:

Clinical pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined
Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins,Triglycerides,Cholesterol
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.
Parameters and methods of cytological examination in BAL: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase
Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin

Necropsy
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes werelavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage,
the animals were necropsied and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).

The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder


Extend of histological processing and sub-sequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal)

Other examinations:

Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.

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
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.

Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period the animals showed no clinical signs and findings different from normal.
During the exposure period the animals of the control group, showed no clinical signs and findings different from normal.
During the study two of the ten animals of test group 1 (5 mg/m3), seven of the ten animals of the test group 2 (20 mg/m3) and all the ten animals of test group 3 (60 mg/m3 ) showed test item
contaminated fur. Five of the ten animals of test group 3 (60 mg/m3) showed discolored fur up to day 19 of the post-exposure observation period. These findings were considered treatment-related, but not adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects 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, treatment-related

Description (incidence and severity):

The significant increase of absolute (+17.6%) and relative (+14.2%) lung weight in animals of test group 3 (60 mg/m³) is regarded as treatment-related and correlates with histopathological findings. It was reversible within the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of test group 2 and 3 (20 and 60 mg/m³) showed a brown discoloration of the lungs as well as mediastinal and tracheobronchial (test group 3, only) lymph nodes. These findings were regarded to be treatment-related.
After recovery, only animals of test group 3 (60 mg/m³) showed a brown discoloration of the lungs. Animals of the same test group revealed a brown discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the base of the epiglottis of four animals of test group 3 (60 mg/m³) and two animals of test group 2 (20 mg/m³), a minimal focal area with flattened epithelium and loss of cilia was seen. This finding was regarded to be treatment related.

In the lungs an increase of alveolar histiocytes was observed which contained brown-yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³). Within the alveolar septae the same particles were observed either within pneumocytes, within histiocytes
located inside the septae and/or free within the interstitium. Additionally, animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to slight hyperplasia of pneumocyte type II cells, a hyperplasia/hypertrophy of bronchioli and a minimal to slight infiltration of intraalveolar neutrophils. Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen. Inside blood vessels occasionally single particle laden macrophages were observed. In test group 3 only, intraalveolar cellular debris was seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above mentioned brown-yellow particles within their cytoplasm. These findings were regarded to be treatment-related

Recovery findings:
In general, similar findings were observed in recovery animals as described for main group animals.
Comparable particles within histiocytes as described for the main group animals were observed in treated recovery animals of test groups 2 and 3 (20 and 60 mg/m³). The histiocytes tended to
accumulate in the area of the broncho-alveolar transition. Macrophages with particles within the BALT were observed in animals of test group 2 and 3 (20 and 60 mg/m³) as well as particles
within alveolar septae in test group 3, only. Single macrophages containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³).
In the mediastinal and tracheobronchial lymph nodes of test group 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed comparable particles as described for the lungs. In test group 3 animals (60 mg/m³) an increase of agglomerated macrophages with particles was seen. These findings were regarded to be treatment-related.
One animal of test group 3 (60 mg/m³) revealed a minimal epithelial alteration in the larynx (levelI).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid: At the end of the administration period, absolute and relative neutrophil counts as well as absolute monocyte counts were increased in BAL in males of test groups 2 and 3 (20 and 60
mg/m3). In addition, males of test group 3 showed increased absolute lymphocyte and epithelial cell counts. These changes led to a concurrent significant increase in the total cell counts in
BAL in test groups 2 and 3, whereas relative macrophage counts were slightly, but significantly decreased. These alterations were regarded as treatment-related and adverse.
At the end of the administration period in BAL of males in test groups 2 and 3 (20 and 60 mg/m3), total protein levels (MTP BAL) as well as -Glutamyl-transferase (GGT BAL), lactate dehydrogenase (LDH BAL), and alkaline phosphatase (ALP BAL) activities were significantly
increased. Further, N-Acetyl-β-D-glucosaminidase (NAG BAL) activities were also significantly increased in males of test group 3. These changes were regarded as treatment-related and adverse.

At the end of the administration period, osteopontin levels were significantly increased in BAL of males in test groups 2 and 3 (20 and 60 mg/m3 ). This alteration was regarded as treatment-related and adverse.
After the three-week recovery period, absolute and relative neutrophil counts were still marginally increased, while relative macrophage counts were marginally decreased in BAL of males in test group 3 (60 mg/m3). Additionally, cytokine-induced neutrophil chemoattractant-1levels (IL-8 BAL) were marginally, but significantly increased. These findings were considered treatment-related and adverse.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal and tracheobronchial lymph nodes of all test groups macrophages were observed that revealed the same brown-yellow particles as described for the lungs. These findings were regarded to be treatment-related.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: particle clearance by macrophages observed, which is the physiological process to remove particles and not an adverse reaction.

2

Dose descriptor:

NOEC

Remarks:

systemic effects

Effect level:

>= 60 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: only findings in BALF

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

20 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Table1: Test concentration and particle size distribution

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		MMAD (µm)	GSD	Fraction of particles < 3 µm [%]
		Mean	SD
01	5.0	5.1	0.6	0.83 0.73	2.96 3.07	88.1 89.7
02	20.0	20.1	0.2	1.05 1.02	2.78 2.77	84.8 85.4
03	60.0	59.7	2.4	0.74 0.90	3.02 2.81	89.7 87.8

SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter. The SMPS showed very high particle count concentrations in all concentrations. The count concentration increased with ascending concentration. The geometric mean count diameters were between 353 nm and 373 nm. There were no difference between the test groups.

Table 2: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 25 (3 weeks after last exposure).

Analyte	Study day 5			Study day 25
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3		Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3
Total Cells	1.0	2.1*	6.0**		1.0	0.8	1.1
Eosinophils	1.8	6.9	9.8		0.7	0.0	0.6
Lymphocytes	0.7	2.5	15.0*		0.4	0.5	1.3
Macrophages	1.0	0.8	1.3		1.0	0.8	0.9
Neutrophils	2.0	67.3**	310.8**		0.8	1.5	3.5*
Monocytes	2.5	3.0*	21.4*		0.5	0.5	2.2
Epithelial cells	1.4	0.7	3.0*		0.7	0.3	0.8

One-sided Wilcoxon-test: *: p<=0.05; ** : p<=0.01

Table 3 Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 25 (3 weeks after last exposure)

Analyte	Study day 5			Study day 25
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3
Total Protein	1.1	2.0**	3.3**	1.0	0.9	1.0
GGT BAL	1.2	3.1**	4.5**	1.2	0.9	1.3
LDH BAL	1.0	2.1**	4.8**	1.2	1.1	1.6
ALP BAL	1.0	2.8**	3.4**	1.1	1.0	1.1
NAG BAL	1.2	1.6	1.9**	0.6	0.4	0.9

GGT =g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = N-Acetyl-β-D-glucosaminidase, One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01

Table 4:  Incidence of gross lesions in main group animals

	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	3	5
Mediastinal lymph nodes
·       Discoloration	0	0	2	4
Tracheobronchial lymph nodes
·       Discoloration	0	0	0	1

Table 5: Incidence and severity of histological findings in the larynx of main group animals

Larynx (level I)	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Alteration, epithelial, (m)f	0	0	2	4
Grade 1			2	4

Table 6:  Incidence and severity of histological findings in the lungs of main group animals

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia type II pneumocytes, (m)f	0	0	5	5
·       Grade 1			5
·       Grade 2				5
Hyperplasia/hypertrophy, bronchioli, (m)f	0	0	4	5
·       Grade 1			4
·       Grade 2				5
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5
Grade 2				5
Particles within histiocytes*	0	5	0	0
Infiltrate neutrophilic, (m)f	0	0	5	5
·       Grade 1			5
·       Grade 2				5
Particles, alveolar septae, (m)f	0	0	3	5
·       Grade 1			3	1
·       Grade 2				4
Macrophages with particles, i.v.	0	0	1	4
·       Grade 1			1	4
Debris, cellular, (m)f	0	0	0	5
·       Grade 1				5
Balt: macrophages with particles (m)f	0	1	2	5
·       Grade 1		1	2	5

Table 7: Incidence and severity of histological findings in the mediastinal lymph nodes of main group animals

Mediastinal lymph nodes	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles, (m)f	0	1	5	5
Grade 1		1	4
Grade 2			1	5

Table 8: Incidence of gross lesions in recovery group animal

	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	2
Mediastinal lymph nodes
·       Discoloration	0	0	0	5

Table 9: Incidence and severity of histological findings in the lungs of recovery group animals

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia type II pneumocytes, (m)f	0	0	0	5
·       Grade 1				5
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5	1
Grade 2				4
Particles within histiocytes*	0	5	0	0
Infiltrate neutrophilic, (m)f	0	0	0	5
·       Grade 1				5
Particles, alveolar septae, (m)f	0	0	0	3
·       Grade 1				3
Macrophages with particles, i.v.	0	0	0	4
·       Grade 1				4
Balt: macrophages with particles (m)f	0	0	5	5
·       Grade 1			5	5

* was diagnosed as present

Table 10: Incidence and severity of histological findings in the mediastinal lymph nodes of recovery group animals

Mediastinal lymph nodes	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophage aggregates w particles, (m)f	0	0	4	5
Grade 1			4
Grade 2				5
Macrophages with particles, (m)f	0	0	2	0
Grade 1			2

Applicant's summary and conclusion

Executive summary:

Inhalation exposure of rats to 60 mg/m³ Pigment Brown 23 on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and epithelia cell counts in bronchoalveolar lavage. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, absolute and relative lung weights were increased, minimal infiltration of neutrophils in the lungs, hyperplasia of type II pneumocytes, cellular debris, was observed in the lungs, as well as hyptrophy/hyperplasia of bronchioli. Particles were observed within alveolar septea in the lungs. Many of these findings were also observed at 20 mg/m³ with reduced severity. The effects did not resolve completely at 60 mg/m³ after 3 weeks post-exposure period. Those observed at 20 mg/m³ resolved completely within the postexposure period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ was the lung and the tracheobronchial lymph nodes.