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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2021 - July 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

Constituent 1
Chemical structure
Reference substance name:
Diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]
EC Number:
275-639-3
EC Name:
Diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]
Cas Number:
71566-54-6
Molecular formula:
C50 H42 Cl2 N6 O8
IUPAC Name:
diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation:
- Particle size distribution:
- Mass median aerodynamic diameter (MMAD):
- Geometric standard deviation (GSD):
- Shape of particles:
- Surface area of particles: BET = 29.5 +/- 0.3 m2/g
- Coating: none

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):
>= 0.51 - <= 0.77 µm
Geometric standard deviation (GSD):
3.5
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 / concentrationsopen allclose all
Dose / conc.:
4.9 mg/m³ air (analytical)
Dose / conc.:
19.8 mg/m³ air (analytical)
Dose / conc.:
60 mg/m³ air (analytical)
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
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:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related

Effect levels

open allclose all
Dose descriptor:
LOAEC
Remarks:
local effects
Effect level:
5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
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.:
5 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: Test concentration and particle size distribution

Test group Target concentration Measured concentration (mg/m³) MMAD (µm) GSD Fraction of particles
(mg/m³) Mean SD < 3 µm [%]
1,00 5,00 4,90 0,70 0,51 3,92 90,30
0,62 3,67 88,80
2,00 20,00 19,80 1,30 0,58 3,41 91,00
0,77 2,90 89,60
3,00 60,00 60,00 2,90 0,67 3,49 88,50
0,59 3,67 89,40

Applicant's summary and conclusion

Conclusions:
Inhalation exposure of rats to 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte counts in bronchoalveolar lavage, while relative macrophage count was reduced in lavage fluid. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as hyptrophy/hyperplasia of terminal bronchi (terminal and small),hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. These findings were also observed at 20 mg/m³ and 5 mg/m³ with reduced severity.
 
After the post-exposure period of 3 weeks, the effects resolved only partly in the mid and high dose group whereas in the low dose group reversibility was complete.
 
Under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could not be determined. The systemic NOAEC is above 60 mg/m³ (high concentration group).
Executive summary:

The following substance-relative adverse findings were observed:

  

Test group 3 (60 mg/m³), main groups

  •  Increase of absolute and relative lung weights (+31.1%/+32.3%)
  • Macroscopically red discoloration of the lungs in all animals
  •  Slight alveolar histiocytosis with red particles in the lungs of all animals
  •  Minimal to slight infiltration of neutrophils in the lungs of all animals
  • Minimal to slight cellular debris in the lungs of all animals
  •  Slight to moderate numbers of particles within bronchi/interstitium in the lungs of all animals
  • Moderate to severe hypertrophy/hyperplasia of bronchi (terminal and small) in the lungs of all animals
  •  Slight hyperplasia of type II pneumocytes in the lungs of all animals
  •  Minimal to slight increased cellularity in the mediastinal lymph nodes in 4 out of 5 animals
  • Increasedtotal protein levels as well asg-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and N-acetyl-β-D-glucosaminidase (NAG) activities in BAL
  • Increased cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8), monocyte chemoattractant protein-1 (MCP-1) and osteopontin levels in BAL
  • Increased total cell counts as well as absolute and relative neutrophil, monocyte and eosinophil cell counts in BAL
  • Increased absolute lymphocyte and macrophage counts in BAL
  • Decreased relative macrophage counts in BAL

Test group 2 (20 mg/m³), main groups

  •  Increase of absolute and relative lung weights (+12.2%/+18.7%)
  •  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 cellular debris in the lungs of all animals
  •  Slight numbers of particles within bronchi/interstitium in the lungs of all animals
  •  Slight to moderate hypertrophy/hyperplasia of bronchi (terminal and small) in the lungs of all animals  Minimal to slight hyperplasia of type II pneumocytes in the lungs of all animals
  •  Minimal to slight hyperplasia of type II pneumocytes in the lungs of all animals
  • Increased total protein levels as well asg-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and N-acetyl-β-D-glucosaminidase (NAG) activities in BAL

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

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

  • Increased absolute lymphocyte and eosinophil cell counts in BAL

  • Decreased relative macrophage counts in BAL

Test group 1 (5 mg/m³), main groups

  •  Macroscopically red discoloration of the lungs in all animals
  •  Minimal alveolar histiocytosis with red particles in the lungs of all animals
  •  Minimal infiltration of neutrophils in the lungs of 4 out of 5 animals
  • Minimal numbers of particles within bronchi/interstitium in the lungs of all animals
  •  Minimal to slight hypertrophy/hyperplasia of bronchi (terminal and small) in the lungs of all animals
  • Increasedtotal protein levels as well asg-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and N-acetyl-β-D-glucosaminidase (NAG) activities in BAL

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

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

  • Increased absolute lymphocyte and eosinophil cell counts in BAL
  • Decreased relative macrophage counts in BAL

Test group 3 (60 mg/m³) after 3 weeks post-exposure observation period

  •   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 2 out of 5 animals
  •  Slight numbers of particles within bronchi/interstitium in the lungs of all animals
  • Minimal hypertrophy/hyperplasia of bronchi (terminal and small) in the lungs of 3 out of 5 animals
  •  Minimal to slight hyperplasia of type II pneumocytes in the lungs of all animals
  •  Minimal to slight increased cellularity in the mediastinal lymph nodes in 4 out of 5 animals

  • Increased lactate dehydrogenase (LDH) activities in BAL

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

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

  • Increased relative neutrophil cell counts in BAL

  • Decreased relative macrophage counts in BAL

 

Test group 2 (20 mg/m³) after 3 weeks post-exposure observation period

  •  Increased lactate dehydrogenase (LDH) activities in BAL

  • Increased cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8), monocyte chemoattractant protein-1 (MCP-1) and osteopontin levels in BAL
  •  Increased total cell counts as well as absolute neutrophil and monocyte counts in BAL

Test group 1 (5 mg/m³) after 3 weeks post-exposure observation period

No treatment-related, adverse effects