N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)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:

March 2022 - Sept 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'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

223-460-6

EC Name:

N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

3905-19-9

Molecular formula:

C40H24Cl4N6O4

IUPAC Name:

N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Test material form:

solid

Specific details on test material used for the study:

IUPAC-Name: N,N'-Phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
Pigment Red 166
Batch 0004846412
Purity: >99%
red solid
storage: room temperature
storage stability: 18 Jan 2023

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 (when supplied)
- Weight at study initiation (means):ca 280g
- 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


IN-LIFE DATES: From: 15 March 2022 (arrival of animals in testing facility) To: 04 April 2022

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.56 - <= 0.73 µm

Geometric standard deviation (GSD):

2.7

Remarks on MMAD:

All measurements of particle size resulted in MMADs between 0.56 and 0.73 µm with GSDs between 2.16 and 3.47.
The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 91.2 % and 97.6 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

SMPS showed different geometric mean concentration than those measured by cascade impactor measurement (see attached figure). 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 345 nm and 367 nm. There was no difference between the test groups.

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.:

4.9 mg/m³ air (analytical)

Remarks:

60049 particles per cm3 (geometric mean diameter 345 nm)

Doses / concentrations 2

Dose / conc.:

20 mg/m³ air (analytical)

Remarks:

187917 particles per cm3 (geometric mean diameter 361 nm)

Doses / concentrations 3

Dose / conc.:

60.4 mg/m³ air (analytical)

Remarks:

289774 particles per cm3 (geometric mean diameter 357 nm)

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: Effective concenrations in same type of study with other 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 and once a day on Saturday and Sundays.

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 will be 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 exposure period all animals of the mid and high concentration (20 and 60 mg/m³) showed substance-contaminated fur, as well as 9 of the 10 animals exposed to the low concentration (5 mg/m³). Moreover, substance-like discoloration of the fur (entire body) was observed in all animals of the high concentration of the test substance.
During the post-exposure period, animals of the recovery group that was exposed to the high concentration of the test substance still showed substance-like discoloration of the fur. The discoloration in the head region persisted up to study day 15. This clinical finding was substance-related, but not adverse because it simply shows the exposure to the solid coloured dusty test substance.

No adverse findings were noted.

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):

See table 1 and 2.

Statistically increased absolute and relative organ weights were observed for following organs: Adrenal glands in test group 2 and 3 (20 and 60 mg/m³) and thyroid glands for test group 1 and 2 (5 and 20 mg/m³).
The mean relative heart weight was significantly decreased in group 3 (60 mg/m³).

The significant increase of absolute and relative adrenal weight in animals of test group 2 and 3 (20 and 60 mg/m³) might be treatment-related (stress). The significant decrease of relative heart weight in animals of test group 3 (60 mg/m³) was regarded to be incidental as the absolute heart weight was not significantly changed. Also, the significant increase of absolute and relative thyroid glands weight in test group 1 and 2 (5 and 20 mg/m³) was judged to be incidental due to a missing dose response relationship.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

No significant differences were observed in the absolute and relative mean organ weights when compared to the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See table 3

Findings after 5-days of treatment:
Animals of test group 2 and 3 (20 and 60 mg/m³) showed a red discoloration of the lungs. Animals of test group 3 (60 mg/m³) revealed red discoloration of the mediastinal lymph nodes in addition. These findings were regarded to be treatment-related.
The discoloration of the skin in all animals of the same test group was regarded to be deposition on fur of the test substance while exposure. No finding in light microscopy could be related to this discoloration.

Findings after recovery
Animals of test group 2 and 3 (20 and 60 mg/m³) showed a red discoloration of the lungs. Animals of the same test groups revealed red discoloration of the mediastinal lymph nodes (all animals of the high dose group and one animal of the mid dose group). These findings were regarded to be treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in lungs and draining lymph nodes (mediastinal and
tracheobronchial).
In the main group, at the base of the epiglottis of four animals of test group 3 (60 mg/m³) a minimal focal area with flattened epithelium and loss of cilia was seen. This finding was regarded to be treatment related. Four animals of test group 3 (60 mg/m³) and one animal of the control group showed alteration in the epithelial of the larynx (level I).

Findings in the lungs of the main group and the recover group are shown in Table 4.
In the lungs an increase of alveolar histiocytes was observed which contained red, spheroid 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. Animals of test group 3 (60 mg/m³) revealed a minimal increase of pneumocyte type II cells and a minimal infiltration of neutrophils intraalveolar. 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.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above mentioned red particles within their cytoplasm. These findings were regarded to be treatment-related.
In general, similar findings were observed in recovery animals as described for main group
animals.

In the tracheobronchial and mediastinal lymph nodes macrophages with particles were reported (Table 5). In the tracheobronchial lymph nodes of all test groups macrophages were observed that revealed the same red particles as described for the lungs. These findings were regarded to be treatment-related.
Similar findings in the tracheobronchial and mediastinal lymph nodes were also described for the recovery group. In recovery test group 3 animals (60 mg/m³) an increase of agglomerated macrophages with particles was seen. These findings were regarded to be treatment-related.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL)

After the administration period, in males of test group 3 (60 mg/m3) total cell counts in BAL as well as absolute and relative neutrophil and monocyte counts as well as absolute macrophage and lymphocyte counts were increased (absolute and relative monocyte counts not statistically significantly). Relative macrophage counts in BAL were significantly decreased. In BAL of males of test group 2 (20 mg/m3) absolute and relative neutrophil counts were already relevantly increased although not statistically significantly. These changes were regarded as treatment related and adverse.
In BAL of males in test group 1 and 2 (5 and 20 mg/m3) epithelial cell counts were increased (in test group 2 not statistically significantly), but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
After the three-week recovery period all altered cell counts values in BAL were back in the normal range.

After the administration period in BAL of males in test group 3 (60 mg/m3) total protein values as well as lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) activities were significantly increased. The same was true for GGT activities in BAL of males in test group 2 (20 mg/m3). These alterations were regarded as treatment related and adverse.
In BAL of males in test group 2 (20 mg/m3) ALP activities were already significantly increased, but the change was only marginal (below 2-fold). Therefore, this alteration was regarded as treatment related but non-adverse.
After the three-week recovery period, all BAL parameter values were back in the normal range.


Cytokines in BAL
After the administration period, in males of test group 3 (60 mg/m3) CINC-1/IL-8 and osteopontin levels in BAL were significantly increased. CINC-1/IL-8 levels were already significantly increased in males of test group 2 (20 mg/m3). These alterations were regarded as treatment related and adverse.
After the three-week recovery the cytokine levels recovered completely.

Effect levels

1

Dose descriptor:

NOEC

Remarks:

local effects

Effect level:

4.9 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: at 20 mg/m3: Increased absolute and relative neutrophils, increaesed GGT and CINC-1/IL-8 levels in BAL after exposure (reversible after recovery period)

2

Dose descriptor:

NOEC

Remarks:

systemic effects

Effect level:

>= 60.4 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.:

60.4 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

Table 1: Relative changes of absolute organ weights of main group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Adrenal glands	+9.3%	+21.0%**	+28.7%**
Thyroid glands	+24.7%*	+30.9%*	+12.8%

*p <= 0.05; **p <= 0.01

Table 2: Relative changes of relative organ weights of main group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Adrenal glands	+14.3%	+23.7%*	+27.8%**
Heart	+2.0%	-3.4%	-6.8%**
Thyroid glands	+24.7%*	+33.3%*	+11.6%

*p <= 0.05; **p <= 0.01

Table 3: Gross lesions after exposure and after recovery

Gross Lesions (5 males/group)	Main group				Recovery group
	control	5 mg/m3	20 mg/m3	60 mg/m3	control	5 mg/m3	20 mg/m3	60 mg/m3
Lung discoloration	0	0	4	5	0	0	5	4
Mediastinal lymph node discoloration	0	0	0	5	0	0	1	5

 

 

Table 4: Incidence and severity of histological findings in the lungs of main and recovery group animals

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

* was diagnosed as present

 

 

Table 5: Incidence and severity of histological findings in the mediastinal lymph nodes of main and recovery group animals

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

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect concentration (NOAEC) for local effects was 4.9 mg/m³ for Pigment Red 166. The systemic NOAEC is above 60.4 mg/m³ (high concentration group).

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 Pigment Red 166 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 nine-week-old male Wistar rats were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 4.9 mg/m³, 20 mg/m³, and 60.4 mg/m³ mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days. Mortality body weight, food consumption, and clinical observations were determined during the study. One half of the rats (5 animals per group) was examined at the end of the exposure period, whereas the other half (5 animals per group) 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 0.56 and 0.73 µm with GSDs between 2.16 and 3.47. The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 91.2 %.

The following substance-relative adverse findings were observed:

 

Test group 3 (60 mg/m³), main group, 1 day after the last exposure

• Increased total cell counts as well as absolute and relative neutrophil counts as well as absolute monocyte, lymphocyte and macrophage counts in BAL

• Decreased relative macrophage counts in BAL

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

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

• Macroscopically red discoloration of the lungs in all animals

• Slight alveolar histiocytosis with red particles in the lungs of all animals

• Minimal infiltration of neutrophils in the lungs of all animals

• Minimal numbers of particles within alveolar septae in the lungs of all animals

• Minimal hyperplasia of type II pneumocytes in the lungs of all animals

 

Test group 3 (60 mg/m³), post-exposure group, 3 weeks after the last exposure

No adverse effects were observed.

 

Test group 2 (20 mg/m³), main group, 1 day after the last exposure

• Increased absolute and relative neutrophil counts in BAL

• Increased γ-glutamyl transferase (GGT) activities in BAL

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

 

Test group 2 (20 mg/m³), post-exposure group, 3 weeks after the last exposure

No adverse effects were observed.

 

Test group 1 (5 mg/m³), main group, 1 day after the last exposure and 3 weeks after last exposure

• No treatment-related, adverse effects were observed