Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with p-phenylenediamine and sodium methoxide

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:

1 Oct 2019 - 04 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

COMMISSION REGULATION (EU) No 260/2014 of 24 January 2014

Deviations:

not specified

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group (Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]

EC Number:

226-999-5

EC Name:

3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]

Cas Number:

5590-18-1

Molecular formula:

C22H6Cl8N4O2

IUPAC Name:

3,3'-(1,4-phenylenediimino)bis(4,5,6,7-tetrachloro-1H-isoindol-1-one)

Constituent 2

Reference substance name:

mono/bis-methoxy derivatives

Molecular formula:

C22-24H6-12Cl6-8N4O2-4

IUPAC Name:

mono/bis-methoxy derivatives

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance under storage conditions over the test period was guaranteed until 29 December 2027 by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: Please, see section "Details on inhalation exposure"

FORM AS APPLIED IN THE TEST: dust aerosol

OTHER SPECIFICS
- physical state: solid
- color: orange

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat was the most frequently used laboratory animal, and there is extensive experience with this species. The rat is also proposed as a suitable test animal by OECD and the EPA. The Wistar strain was selected because a huge amount of historical control data was available for this strain. Furthermore, concerning pulmonary lavage in rats there is a considerable database for this species available in published literature (Henderson 1984, Henderson et al., 1985, Driscoll et al., 1990, Warheit et al., 1991 and 1995) and rats were sensitive in pulmonary toxicity studies.

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, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation (mean): 307g (on day -3)
- Fasting period before study: not specified
- Housing: The rats were housed together (up to 5 animals per cage)
- Diet: Mouse/rat laboratory diet; ad libitum
- Water: tap water, ad libitum
- Acclimation period: about 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- The food used in the study was assayed for chemical as well as for microbiological contaminants. On the basis of the duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants as well as for bacteria. On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 – 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark)

IN-LIFE DATES: From: 01 Oct 2019 (Arrival) To: 24 Oct 2019

Administration / exposure

Route of administration:

other: inhalation: dust aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.23 - <= 1.87 µm

Geometric standard deviation (GSD):

2.28

Remarks on MMAD:

The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 70 %.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-onlyaerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The exposure systems were located in exhaust hoods in an air conditioned room.
- Method of holding animals in test chamber: rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Source and rate of air: Compressed air was produced by an oil-free compressor. For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter, the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: The central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter. The so generated conditioned air was used to generate inhalation atmospheres.
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass dilution tube, Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. 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.
- Temperature, humidity, pressure in air chamber: The daily mean relative humidities in the inhalation systems ranged between 38.9 and 60.2 %. The daily mean temperatures in the inhalation systems ranged between 22.2 and 23.4°C. Compressed air was filtered air pressurized to about 5-6 bar.
- Air flow rate and Air change rate were recording with the automated measuring system. The air flows were constantly maintained in the desired range. An air change between 66 and 68 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: gravimetrical measurement
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
Brief description of analytical method used:
- Calulation of nominal concentrations: The nominal concentration was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
- Analytical determination of concentrations: The concentrations of the inhalation atmospheres were determined by gravimetric measurement of filter samples in test groups 1 to 3. In these groups, the constancy of concentrations in each inhalation system was continuously monitored using scattered light photometers. In the control group (test group 0) no samples were taken.
- Particle size analysis was carried out with a cascade impactor.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures. During some daily exposures oscillations and higher variability occurred in the mid and high concentration. The reason for this behaviour could not be identified, because generation and exposure procedure were not changed all over the study. It was not reflected in the analytically determined concentrations, which integrate the variations over the sampling time.
Please see table 6 in section "Any other information on materials and methods incl. tables"

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, for five consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 mg/m³ air

Doses / concentrations 2

Dose / conc.:

10 mg/m³ air

Doses / concentrations 3

Dose / conc.:

30 mg/m³ air

No. of animals per sex per dose:

5/dose/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not specified
- Fasting period before blood sampling for clinical biochemistry: Before blood sampling and necropsy food will be withdrawn from the animals overnight (about 16-20 hours).
- Rationale for selecting recovery groups: not specified (random)
- Post-exposure recovery period : In order to check for any reversibility, progression or delay of toxic effects the following post-exposure observation groups were used, these being observed for about 3 weeks after the exposure period (5 animals per group).
- Other: By using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is with about 4 min shorter as compared to whole-body chambers with a higher chamber volume.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

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

BODY WEIGHT: Yes
- Time schedule: The animals were weighed prior to the pre-exposure period (study day -3), 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, 9, 16, 23 and 25.
The body weight change was calculated as the difference of actual body weights and the
weights of last weighing.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure), in the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/group/time point
- Parameters checked in table 1 were examined (please see section "Any other information on materials and methods incl. tables")

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure), in the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Animals fasted: Yes
- How many animals: 5/group/time point
- Parameters checked in table 2 were examined (please see section "Any other information on materials and methods incl. tables")

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure). The animals 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.
- Dose groups that were examined: All dose groups and the control group
- Number of animals: 5/group/time point
- Parameters checked in table 3 and 4 were examined (please see section "Any other information on materials and methods incl. tables")
- Antigens measured in BALF by ELISA: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Macrophage colony stimulating factor (M-CSF), Rodent osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. After the lavage procedure, the left and right lung lobes were instilled with formalin for weight assessment and further histopathological assessment. Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

GROSS PATHOLOGY: Yes
- Organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands. All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
- Organ/tissue fixation: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder.
Fixation was followed by histotechnical processing, examination by light microscopy and
assessment of findings according to the table 5 (plese see section "Any other information on materials and methods incl. tables").

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters.
- 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.
- Broncho-alveolar lavage fluid (BALF): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Weight parameters in pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity in animals of control and test group 1 through out the study period. In test group 2 (10 mg/m³), substance contaminated fur was observed on study day 3 and day 4 after exposure in one main group and one recovery group animal, respectively. In test group 3 (30 mg/m³), substance contaminated fur was observed for 7 animals on study day 2 after exposure, for 2 animals on study day 3 after exposure and for 4 animals on study day 4 after exposure. No other signs of toxicity were observed in test groups 2 and 3.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0 through out the whole study period. The mean body weight change of the animals exposed to the test substance up to the highest concentration of 30 mg/m³ were comparable to the concurrent control animals throughout the study period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At study day 5, in males of test group 2 (10 mg/m3) platelet counts were significantly increased, but the alteration was not dose dependent. Therefore, it was regarded as incidental and not treatment related. After a 3-week recovery, in males of test group 1 (3 mg/m3) mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. Additionally, in males of test group 2 (10 mg/m3) MCH was significantly lower compared to controls. These changes were not dose dependent. In males of test groups 2 and 3 (10 and 30 mg/m3) relative basophil counts were increased (in test group 3 not statistically significantly), but the values were within the historical control range (males, relative basophils 0.1-0.6 %). Therefore, these alterations were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At study day 5, in males of test groups 2 and 3 (10 and 30 mg/m3) globulin values were decreased (in test group 3 not statistically significantly), but the values were within the historical control range (males, globulins 21.58-29.13 g/L). Therefore, this alteration was regarded as incidental and not treatment related. After the 3-week recovery, in males of test group 2 (10 mg/m3) total protein, globulin, glucose and cholesterol values were significantly decreased. However, the values were not dose dependently changed. Therefore, these alterations were regarded as incidental and not treatment related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, all mean absolute and relative weight parameters did not show significant differences when compared to the control group.
- In the recovery group, the mean relative kidney weight of males in test group 1 was significantly increased, however this change was not related to the exposure concentration and was considered as incidental and not treatment-related. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, two males in test group 2 had foci within the epididymis. These findings occurred individually and were therefore considered to be incidental or spontaneous in originband without any relation to treatment.
- In the recovery group, one male in test group 1 had focal liver constrictions. This finding occurred individually and was therefore considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Histological findings in the lungs of main group animals: macrophages, which contained a brown to goldish pigment (presumably inhaled test compound)
were multifocally distributed within the lumen of alveoli and terminal bronchioles, with a concentration-dependent increase in number of macrophages and amount of pigment storage within each macrophage, starting from minimal in test group 1 (3 mg/m3) up to mild in test group 3 (30 mg/m3). In test group 1 few pigment particles were within macrophages and therefore could only be visualized at a 100x magnification. No extracellular pigment or signs of inflammation were detected. Single intravascular pigment-laden macrophages were present in animals of all test groups. Furthermore, a minimal number of pigment-laden macrophages were present in the bronchus-associated lymphoid tissue (BALT) in animals of all test groups, with a dose dependent increase in affected animals.
- Histological findings in the lungs of recovery group animals: Pigment-laden macrophages were multifocally distributed in the lumen of the alveoli of all animals of all test groups exposed to the test substance. In general, the number of pigmentladen alveolar macrophages was lower in all exposed recovery group animals compared to exposed animals of the corresponding main groups. In test group 1 (3 mg/m3) macrophages were so inconspicuously laden with pigment that this could only be visualized at 200x. In 3 out of 5 animals of test group 1 (3 mg/m3) the number of alveolar macrophages was not increased compared to the control group 0, but the present macrophages contained pigment. The remaining 2 animals of test group 1 (3 mg/m3) and all animals of test group 2 (10 mg/m3) displayed a minimal increase of alveolar macrophages laden with pigment. All animals of test group 3 (30 mg/m3) displayed a minimal to mild increase of pigment-laden alveolar macrophages, in 3 animals the macrophages formed aggregates, which showed a tendency to concentrate in the bronchiolo-alveolar junction. Single intravascular macrophages were present in animals of all test groups. Examination of the bronchus-associated lymphoid tissue (BALT) revealed a minimal to mild number of pigment-laden macrophages in animals of all test groups.

- Histological findings in the mediastinal lymph nodes of main group animals: In test group 3 (30 mg/m3), 2 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages.
- Histological findings in the mediastinal lymph nodes of recovery group animals: In test group 2 (10 mg/m3), 3 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages, whereas in test group 3 (30 mg/m3) in 3 out of 5 animals macrophages formed multifocal aggregates with a minimal to mild severity. For details please see table 5 in section “Any other information on results incl. Tables”.

- Histological findings in the tracheobronchial lymph nodes of main group animals: In test group 3 (30 mg/m3), 3 of 5 animals showed a minimal number of multifocally distributed single pigment-laden macrophages.
- Histological findings in the tracheobronchial lymph nodes of recovery group animals: In test group 2 (10 mg/m3), 1 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages, whereas in test group 3 (30 mg/m3) in 3 out of 5 animals macrophages formed multifocal aggregates with a minimal to mild severity.

- Histological findings in the larynx of main group animals: A minimal epithelial alteration, characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers) was present at level I in 1 animal of test group 1 (3 mg/m3), 1 animal of test group 2 (10 mg/m3) and 3 animals of test group 3 (30 mg/m3). 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.
- Histological findings in the larynx of recovery group animals: A minimal epithelial alteration characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers) was present at level I in 2 animals of test group 2 (10 mg/m3). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

At study day 5, in males of test group 3 (30 mg/m3) absolute neutrophil counts were 13.7-fold higher compared to controls and absolute monocyte counts were increased, whereas total cell counts were not greater compared to controls. Relative neutrophil and monocyte counts were also increased. Therefore, higher neutrophil and monocyte counts may be regarded as a marginal adverse effect. After the 3-week recovery, in males of test group 3 (30 mg/m3) absolute macrophage counts were significantly increased, and in males of test group 1 (3 mg/m3) total cell counts in BAL were significantly higher compared to controls. However, the alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

At study day 5, in males of test group 3 (30 mg/m3) alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) activities were significantly increased. However, ALP mean was within the historical control range and GGT activities were marginally above this range (BAL, ALP 0.25-0.51 μkat/L; GGT 20-64 nkat/L). Therefore, these alterations were regarded as incidental and not treatment related. After the 3-week recovery in males of test group 1 (3 mg/m3) lactate dehydrogenase (LDH) and N-acetyl-β-glucosaminidase (NAG) activities were significantly increased, but the changes were not dose dependent. Therefore, these alterations were regarded as incidental and not treatment related.

At study day 5, in males of test groups 2 and 3 (10 and 30 mg/m3) IL-8/CINC-1 levels were significantly increased. Although these increases were marginal, in combination with significant higher neutrophil counts in the BAL of males of test group 3, it can be regarded as treatment related and adverse, whereas the IL-8/CINC-1 increase in test group 2 was isolated and therefore, it is regarded as treatment related but non-adverse. At study day 5 in males of test group 1 (3 mg/m3) osteopontin values were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

For details, please see table 1-3 in section "Any other information on results incl. tables"

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

10 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: findings in lavage fluid

2

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study days 26 (3 weeks after last exposure). At study day 5, mean of differential cell counts in control group (test group 0) is based on only two individuals due to bad cell quality. Therefore, for these parameters, statistics could not be performed.

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
Total Cell count	0.9	0.9	0.9	1.8*	1,2	1.4
Eosinophils	0.0	4.9	0.5	0.8	0.0	0.0
Lymphocytes	1.0	0.3	0.0	+	+	+
Macrophages	0.7	0.7	0.6	1.9	1.2	1.4*
Neutrophils	1.2	0.6	13.7	0.7	1.9	1.2
Monocytes	+	+	+	+	+	+
Epithelial cells	0.0	0.1	0.0	+	+	+

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

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

 

Table 2: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure)

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
Total Protein	1.0	1.0	0.9	1.2	0.9	0.8
GGT	1.0	1.1	1.5**	1.4	1.0	1.2
LDH	0.6	0.9	0.6	1.9*	1.1	1.3
ALP	1.1	1.0	1.3*	0.9	1.1	0.9
NAG	0.7	0.8	0.7	3.2**	1.0	1.9

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = N-Acetyl-β-glucosaminidase

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

 

Table 3: Changes in antigen levels in BAL (x-fold of concurrent control means) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
MCP-1	1.0	1.0	1.1	0.8	0.8	0.7
CINC-1/IL-8	1.3	1.5*	1.8*	0.6	0.9	0.9
M-CSF	1.0	1.0	1.0	1.0	1.0	1.0
Osteopontin	2.1*	1.1	1.4	0.5	0.6	0.5

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

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

Applicant's summary and conclusion

Conclusions:

Inhalation exposure of rats to 30 mg/m³ of the test substance on 5 consecutive days caused Increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in bronchoalveolar lavage. There was no histological correlate. All effects were reversible within 3 weeks exposure-free recovery period. Based on the findings in lavage fluid, the no observed adverse effect concentration (NOAEC) for local effects was 10 mg/m³ under current study conditions. The systemic NOAEC is above 30 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 the test substance 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 nine week old male Wistar rats (10 rats per concentration group) were noseonly exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) on 5 days for 6 hours per day. Body weight, mortality and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and histopathological changes in the target organs. During the exposure period the target concentrations were reached.

The particle size resulted in MMADs between 1.23 and 1.87 μm with GSDs between 2.08 and 2.45. The calculated mass fractions of particles below 3 μm aerodynamic size was greater than 70 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Main group:

- Test group 3 (30 mg/m³): Increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in BAL

- Test group 2 (10 mg/m³), test group 1 (3 mg/m³): No treatment-related adverse effects

Recovery group:

- Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³): No treatment-related adverse effects