3,3'-[(2,5-dichloro-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(5-chloro-o-tolyl)benzamide]

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:

Apr 22 - Oct 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:

3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]

EC Number:

279-356-6

EC Name:

3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]

Cas Number:

79953-85-8

Molecular formula:

C55H37Cl5F6N8O8

IUPAC Name:

3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis{4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide}

Test material form:

solid: nanoform, no surface treatment

Specific details on test material used for the study:

yellow powder

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 265g
- 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):

>= 0.66 - <= 0.77 µm

Geometric standard deviation (GSD):

245

Remarks on MMAD:

All measurements of particle size resulted in MMADs between 0.66 and 0.77 µm with GSDs between 2.06 and 2.90. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 90.0 % and 97.6 %.

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 geometric mean count diameters were between 266 nm and 295 nm.

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.:

4.98 mg/m³ air (analytical)

Remarks:

SD 0.27

Doses / concentrations 2

Dose / conc.:

20 mg/m³ air (analytical)

Remarks:

SD 1.0

Doses / concentrations 3

Dose / conc.:

60.4 mg/m³ air (analytical)

Remarks:

SD 2.1

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:

no effects observed

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

Description (incidence and severity):

In males of test group 3 (60 mg/m3) absolute neutrophil counts were significantly higher compared to controls. The values were slightly above the historical control range (males, neutrophils 0.53-1.01 Giga/L). however, total white blood (WBC) counts as well as all other differential blood cell fractions were not changed. Therefore, this isolated alteration of absolute neutrophil counts in males of test group 3 was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).

After the three-week recovery period, no changes of absolute neutrophil counts were observed. In males of test group 2 (20 mg/m3) absolute and relative, large unstained cell (LUC) counts were significantly higher compared to study controls, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.

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 and relative lung weight in animals of test group 3 (60 mg/m³) and the relative lung weight in test group 2 (20 mg/m³) is regarded as treatment-related and correlates with histopathological findings.
(See tables below). Findings were reversible within the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two animals of test group 3 (60 mg/m³) showed a yellow discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related.
After recovery, three animals of test group three showed a yellow discoloration, and so did one animal of the low dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the lungs animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to moderate hyperplasia/hypertrophy of bronchioli (mainly small and terminal bronchi and the bronchio-alveolar transition were affected). Furthermore, an increase of alveolar histiocytes was observed which contained yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³) and a minimal infiltration of intra alveolar neutrophils in test group 3 animals (60 mg/m³). Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above-mentioned yellow particles within their cytoplasm. These findings were regarded to be treatment-related.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes. In the mediastinal lymph nodes of test groups 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed the same yellow particles as described for the lungs. These findings were regarded to be treatment-related.


Recovery findings:
Comparable to the main groups yellow particles within minimally increased numbers of histiocytes 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 bronchio-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. Alveolar histiocytes containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³) but were not increased in number.
A single animal (No. 39) of test group 3 (60 mg/m³) still showed hyperplasia/hypertrophy bronchiole, neutrophilic infiltrates and cellular debris intra alveolar in addition. It was regarded to be degenerating histiocytes as also free yellow particles were observed. All these findings were regarded to be treatment-related. The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal lymph nodes of all treated test groups macrophages were observed that revealed comparable particles as described for the lungs. They were also found as agglomerated macrophages with particles. 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 examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL)
After the administration period in BAL of males of test group 3 (60 mg/m3) total cell counts as well as above all absolute and relative neutrophil cell and absolute eosinophil cell counts were increased (absolute eosinophil counts not statistically significantly). Additionally, a moderate increase of absolute and relative lymphocyte as well as absolute macrophage, monocyte and epithelial cell counts could be observed. In BAL of males of test group 2 (20 mg/m3) total cell counts were not changed, but absolute and relative neutrophil, lymphocyte counts as well as absolute monocyte cell counts were already increased. Relative macrophage counts in BAL of males in test groups 2 and 3 were decreased. These alterations were regarded as treatment related and adverse.

In males of test group 2 (20 mg/m3) absolute eosinophil counts were also increased, but not statistically significantly. However, the mean value was within the historical control range (males, absolute eosinophils 0.00-0.21 cn/µL BAL). Therefore, this change was regarded as incidental and not treatment related.
After the three-week recovery period all altered cell count values in BAL were back in the normal range. In males of test group 11 (5 mg/m3) relative lymphocyte counts were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

In BAL of males in test group 3 (60 mg/m³) N-acetyl--D-glucosaminidase (NAG) activities were also significantly increased, but the values were within the historical control range (males, NAG 10-79 nkat/L). The same was true for significantly increased total protein levels in BAL of males in test group 2 (20 mg/m3) as well as in GGT activities in BAL of males in test group 1 (5 mg/m3)( males, total protein 18-50 mg/L, GGT 25-64 nkat/L). Therefore, these changes were regarded as incidental and not treatment related.
After the three-week recovery period, all BAL parameter values were back in the normal range.

During the exposure period the animals all animals of the mid and high concentration (20 and 60 mg/m³) showed substance-contaminated fur. Moreover, substance-like discoloration of the fur was observed in all animals of the high concentration of the test substancewerewed substance-like discoloration of the fur on study day 6.

Effect levels

1

Key result

Dose descriptor:

NOAEC

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

2

Key result

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

>= 60 mg/L air (analytical)

Based on:

test mat.

Sex:

male

Remarks on result:

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

Target concentration	Measurement	MMAD (µm)	GSD	Fraction < 3 µm
5 mg/m³	1	0.77	2.90	90.0 %
	2	0.70	2.41	95.0 %
20 mg/m³	1	0.71	2.22	96.4 %
	2	0.66	2.67	93.9 %
60 mg/m³	1	0.72	2.06	97.6 %
	2	0.85	2.51	91.6 %

 

Table 2: Particle count distribution measured by SMPS

Target concentration	Total count concentration (N/cm³)	Geometric mean diameter (nm)	Geometric standard deviation
5	103723	266	1.75
20	340464	295	1.75
60	840216	288	1.75

 

 

Table 3 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 30 (3 weeks after last exposure).

Analyte	Study day 5			Study day 30
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3		Gr. 11 5 mg/m^3	Gr. 12 20 mg/m^3	Gr. 13 60 mg/m^3
Total Cells	1.1	1.2	3.3**		0.6	1.1	0.7
Eosinophils	3.1	10.4	24.4		0.1	0.3	0.4
Lymphocytes	2.0	2.8*	7.9**		1.5	1.0	1.0
Macrophages	1.1	1.1	2.3**		0.6	1.1	0.7
Neutrophils	0.7	5.6**	27.8**		1.3	1.3	2.1
Monocytes	1.3	3.1*	9.9**		8.9	0.0	2.8
Epithelial cells	0.8	0.4	4.1*		0.9	2.4	0.2

One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01: + increase could not be calculated because of zero activity in controls

 

Table 4: 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 30 (3 weeks after last exposure)

 

Analyte	Study day 5			Study day 30
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3	Gr. 11 5 mg/m^3	Gr.2 2 20 mg/m^3	Gr. 13 60 mg/m^3
Total Protein	1.0	1.3*	1.9**	1.3	1.4	1.1
GGT	1.2**	2.4**	3.9**	1.0	1.3	1.0
LDH	1.0	1.1	2.3**	0.9	1.3	1.0
ALP	1.2	1.9**	2.8**	1.1	1.0	1.2
NAG	1.2	1.3	2.2*	1.1	1.3	0.9

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

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

 

Table 5: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)

Analyte	Study day 5			Study day 30
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3	Gr. 11 5 mg/m^3	Gr. 12 20 mg/m^3	Gr. 13 60 mg/m^3
CINC-1/IL-8	1.5*	4.1**	4.2**	1.1	1.4*	1.3
Osteopontin	1.1	1.1	4.3**	1.3	1.4	2.5*

.BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1

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

 

Table 6a: Absolute and relative organ weights (main group)

Test group (mg/m³)	01 (5)	02 (20)	03 (60)
Lungs, absolute (and relative)	+8.8% (+ 3.3%)	+20.9% (+12.6%)*	+24.6%* (+21.8%)*

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

 

Table 6b Relative changes of absolute organ weights (recovery group).

	Male animals
Test group (mg/m³)	11 (5)	12 (20)	13 (60)
Brain	+8.8%**	+4.8%**	+3.5%
Heart	+18.1%**	+5.8%	+15.0%*
Spleen	+18.6%	+11.6%	+34.5%**

NB All mean relative weight parameters did not show significant differences when compared to the control group

 

 

Table 7a: Histopathology ( Incidence and severity of histological findings in the lungs of main group animals)

Lungs	Male animals
Test group (mg/m³)	00 (0)	01 (5)	02 (20)	03 (60)
No. of animals	5	5	5	5
Hyperplasia/hypertrophy, bronchioli, (m)f	0	0	5	5
·       Grade 1			3	1
·       Grade 2			2	2
·       Grade 3				2
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5
Grade 2				5
Particles within histiocytes*	0	5	0	0
Balt: macrophages with particles (m)f	0	0	4	4
·       Grade 1		0	4	4
Infiltrate neutrophilic, (m)f	0	0	0	4
·       Grade 1				4

 

 

Table 7b Incidence and severity of histological findings in the lungs of recovery group animals

Lungs	Male animals
Test group (mg/m³)	01 (0)		11 (5)		12 (20)		13 (60)
No. of animals	5		5		5		5
Hyperplasia/hypertrophy, bonchioli	0		0		0		1
·       Grade 1							1
Debris, cellular, (m)f	0		0		0		1
·       Grade 1							1
Histiocytosis alveolar with particles, (m)f	0		0		5		5
Grade 1					5		5
Macrophages with particles, interstitial (m)f	0		0		0		2
Grade 1							2
Particles within histiocytes*	0		5		0		0
Balt:[WT1] macrophages with particles (m)f	0		0		1		3
·       Grade 1					1		2
·       Grade 2							1
Infiltrate neutrophilic, (m)f	0	0		0		1
·       Grade 1						1

Applicant's summary and conclusion

Conclusions:

Under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3. The systemic NOAEC is above 60 mg/m³ (high concentration group).

Executive summary:

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 and macrophage 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 bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity.

 

After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³. 

 

Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3. The systemic NOAEC is above 60 mg/m³ (high concentration group).