4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

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:

Oct 19, 2021 - Mar 03, 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 25 June 2018

Deviations:

yes

Remarks:

5 days-exposure instead of 28 days

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

24 January 2014

Deviations:

yes

Remarks:

5 days-exposure instead of 28 days

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland Pfalz, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

221-423-9

EC Name:

4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

3089-16-5

Molecular formula:

C20H10Cl2N2O2

IUPAC Name:

4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Batch 200389
- State of aggregation: solid, powder
- The number-based concentration of particles with sizes below 100 nm (smallest feret diameter) is 99%
- Particle size distribution (TEM): 43.3 nm (D50)
- Shape of particles: multimodal/orthorhombic
- Coating: no
- Surface properties: not applicable
- Density: 1689 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable

Test material purity is 100% pigment minus extractables and volatiles

Specific details on test material used for the study:

TEST MATERIAL
- Batch number: 200389
- Purity: 98.4 g/100 g = 100 g/100 g - w (volatile components) - w (extractable components)
- Physical state/ appearance: solid (powder)/ red

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition: room temperature
- Stability: under storage conditions guaranteed until 2030 as indicated by the sponsor, and the sponsor holds this responsibility
- Homogeneity: given visual

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines.
Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: 9 weeks
- Weight at study initiation: weight variation of the animals used did not exceed 20 % of
the mean weight
- Housing: (5 animals per cage) in Typ 2000P ca. 2065 cm² (polysulfone cages) supplied by TECNIPLAST, Germany, bedding in the cages were dust-free bedding, for enrichment wooden gnawing blocks and Play Tunnel were added
- Diet: mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: about 14 days

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

IN-LIFE DATES: From: Nov 02, 2021 To: Nov 10, 2021

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

0.76 - 1.07 µm

Geometric standard deviation (GSD):

3.34

Remarks on MMAD:

All measurements of particle size resulted in MMADs between 0.76 and 1.07 μm with GSDs between 2.73 and 3.34. The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 82.4 and 88.6 %. Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Details on inhalation exposure:

GENERATOR SYSTEMS
- Solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany)
- Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany)
- Glass connection pipe (BASF SE, Ludwigshafen, Germany)

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 tube, mixed with conditioned dilution air and passed into the inhalation system via a glass connection pipe.
- The desired concentrations in the test groups were achieved by adjusting of the piston dosing rate and the brush rotating speed.
- The control group was exposed to conditioned air.
- The following substance flow and air flows were scheduled:
i) test group: 0, substance flow: -, supply air conditioned: 5 - 7 m³/h, supply air compressed: -, exhaust air: 4.5 - 6.5 m³/h
ii) test group: 1, substance flow: 40 - 75 mg/h, supply air conditioned: 4 - 5 m³/h, supply air compressed: 1 - 2 m³/h, exhaust air: 4.5 - 6.5 m³/h
iii) test group: 2, substance flow: 150 - 300 mg/h, supply air conditioned: 4 - 5 m³/h, supply air compressed: 1 - 2 m³/h, exhaust air: 4.5 - 6.5 m³/h
iv) test group: 3, substance flow: 400 - 800 mg/h, supply air conditioned: 4 - 5 m³/h, supply air compressed: 1 - 2 m³/h, exhaust air: 4.5 - 6.5 m³/h
- Conditioned supply air is activated charcoal filtered air conditioned to about 30% to 70% relative humidity and 20°C to 24°C, compressed air is filtered air pressurized to 5 to 6 bar

NOSE-ONLY EXPOSURE SYSTEMS
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V ca. 90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainlesssteel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.

EXPOSURES
The nose-only exposure technique was preferably selected for this dust aerosol inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, 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 minutes shorter as compared to whole-body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on three days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours on each day over a time period suitable to reach 5 exposures. For adaptation to the exposure conditions the male animals were placed into restraining tubes and exposed to conditioned clean air on study day -3, -2 and study day -1, before start of the exposure period (details are available in the raw data). This procedure is referred to as preexposure period. On the first day of the pre-exposure period were exposed to air for 2 hours, on the second day for 4 hours. On the third day they were exposed to air for 6 hours. Then all test groups were exposed to air (control group) or to the test substance (test groups 1 to 3) for 6 hours per day on 5 consecutive days. The animals did not have access to water or food during the exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GENERAL
The concentrations of the inhalation atmospheres in test groups 1 - 3 were determined by gravimetric measurement. 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.
In the control group (test group 0) no sample was measured over the study period.
A pre-weighed filter was placed into the filtration equipment. By means of a vacuum compressed
air pump a defined volume of the dust aerosol was drawn through the filter.
The dust concentration in mg/m³ was calculated from the difference between the weight of the pre-weighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.

EQUIPMENT
- Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA)
- Internal probe diameter: 7 mm
- Filter: MN 85/90 BF (d = 4.7 cm)
- Vacuum pump: Millipore Corporation, Billerica, MA 01821, USA
- Balance: Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)

SAMPLING
- Sampling velocity:
- Flow rate of sampling:
- Sample volumes: test group 1: 180 L, test group 2: 45 L, test group 3: 15 L
- Sampling site: immediately adjacent to the animals' noses at a separate spare port
- Sampling frequency: three samples per exposure and concentration group

REAL TIMR MONITORING OF CONCENTRATIONS
Scattered light photometers (VisGuard (Sigrist) in test groups 1-3 were used to continuously monitor the constancy of concentrations of test substance aerosols. To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were recorded transferred to the automated measuring system.

Duration of treatment / exposure:

5 days

Frequency of treatment:

6 hours per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/m³ air

Doses / concentrations 2

Dose / conc.:

20 mg/m³ air

Doses / concentrations 3

Dose / conc.:

60 mg/m³ air

No. of animals per sex per dose:

5 per main group and 5 per post-exposure observation group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available data, upon approval by the sponsor, the concentrations were selected
- Fasting period before blood sampling for clinical biochemistry: overnight (about 16 - 20 hours)
- Rationale for selecting satellite groups: recovery group animals were examined after an exposure-free period to detect any reversibility or progression of potential toxic effects
- Post-exposure recovery period in satellite groups: 3 weeks

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (in the morning and in the late afternoon) on working days, as well as on Saturday the 6th of November 21 and Sunday the 7th of November 21, on the other Saturdays and Sundays the examinations were performed once a day (in the morning)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 post-exposure observation weekends, no clinical observation was performed

BODY WEIGHT: Yes
- Time schedule: 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, 10, 17and 24

FOOD CONSUMPTION
- 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 during exposure period and in one cage during post-exposure period, no statistical evaluation of food consumption is possible.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after last exposure, in the post-exposure observation group 3 weeks after last exposure
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes
- How many animals: All
- Parameters checked: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular concentration, platelet count, differential blood, reticulocytes

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: after last exposure, in the post-exposure observation group 3 weeks after last exposure
- Animals fasted: Yes
- How many animals: All
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

BRONCHOALVEOLAR LAVAGE FLUID (BAL): Yes
- Time schedule: after last exposure, in the post-exposure observation group 3 weeks after last exposure
- Dose groups that were examined: All
- Number of animals: All
- Parameters checked: total cell count, macrophages, plymorphonuclear neutrophils, lymphocytes, eosinophils, monocytes, epithelial cells, erythrocytes, destruated cells, conglomerated cells, gamma-glutamyltransferase, protein, lactate dehydrogenase. alkaline phosphatase, nacetyl-beta-glucosaminidase, rat cytokine-induced neutrophil chemoattractant-1 level, rodent osteopontin

LUNG BURDEN: Yes
For lung lavage all 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, including weight for anesthetized animals (final body weight) and organ weights of adrenal glands (fixed), brain, epididymides, heart, kidneys, liver, lungs, spleen, testes, thymus (fixed), thyroid glands (with parathyroid glands) (fixed), all paired organs were weighed together (left and right)

HISTOPATHOLOGY: Yes, including all gross lesions, adrenal glands, bone marrow (femur), brain with olfactory bulb, coagulating glands, epididymides, esophagus, eyes with optic nerve, heart, kidneys, larynx/pharynx, liver, lungs, lymph nodes (tracheobronchial and mediastinal lymph nodes), nose (nasal cavity), prostate ,seminal vesicles, spinal cord (cervical, thoracic and lumbar cord), spleen, stomach (forestomach and glandular stomach), testes, thyroid glands, thymus, trachea, urinary bladder

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 p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) 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

Bronchoalveolar lavage fluid (BAL): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

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

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

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure, exposure and post-exposure period the animals of the control group and low concentration (5 mg/m³) group did not show any clinical signs of toxicity. During the exposure period 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 several animals exposed to the mid and the high concentration of the test substance. During the post-exposure period, several animals of the mid and high concentration group showed substance-like discoloration of the fur until study day 7.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the whole study period, the mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0. During the exposure period from study day 0 to 4 the mean body weight change of the male animals of the mid concentration (20 mg/m³) was with -8.4 g significantly (p ≤ 0.05) lower than the controls (-4.0 g). This marginal deviation from the control was considered incidental,
because there was no relation to the exposure concentration.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No statistical evaluation was performed for evaluation of food consumption because the animals were in social housing. During the exposure period, there were two cages (main and postexposure groups), while there was only one cage per concentration during the post-exposure
period. During the exposure period, the food consumption of all test groups was lower than during the post-exposure period. This phenomenon is due to the withdrawal of food and water during daily inhalation exposure. There was no obvious concentration related decrease of food consumption during the exposure period from day 0 to 4.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related, adverse changes among hematological parameters were observed. At the end of the administration period, in males of test group 3 (60 mg/m3) absolute and relative neutrophil counts were increased (absolute neutrophil counts not statistically significantly). Total white blood cell counts, and any other differential blood cell fraction were not altered among these individuals. Therefore, the only changed neutrophil cell counts in males of test group 3 were regarded as maybe treatment related but non-adverse (ECETOC Technical Report No. 85, 2002). In males of test group 2 (20 mg/m³) absolute lymphocyte count were significantly decreased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related. After the three-week recovery period, absolute and relative neutrophil cell counts among males of test group 3 were not changed, anymore. Some other parameter values were altered significantly, but the changes were not dose dependent and therefore, they were regarded as incidental and not treatment related. These changes were: increased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in males of test groups 1 and 2 (5 and 20 mg/m³); Increased absolute basophil cell counts in males of test groups 1 and 3 (5 and 60 mg/m³) as well as increased relative basophil cell counts in males of test groups 1 and 2 (5 and 20 mg/m³).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After the administration period, no treatment related changes among clinical chemistry parameters were observed. After the three-week recovery period, in males of test group 2 (20 mg/m3) total protein values were significantly increased, but the change was not dose dependent. Therefore, this change was 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, treatment-related

Description (incidence and severity):

MAIN GROUP
None of the mean absolute and relative weight parameters showed statistically significant differences when compared to the control group 0. However, the absolute and relative weights of the lungs were dose relatedly increased and changes in absolute weights in the recovery group of a similar magnitude reached statistical significance. When compared to the control group 0 (=100%), the mean absolute and relative weights of the lungs were increased without reaching statistical significance in all treated test groups 1, 2 and 3.

RECOVERY GROUP
When compared to the control group 0 (=100%), the mean absolute weights of the lungs were significantly increased in all treated test groups 1, 2 and 3. All other mean absolute weight parameters did not show significant differences when compared to the control group 0. None of the mean relative weight parameters showed significant differences when compared to the control group 0. However, when compared to the control group 0 (=100%), the mean relative weights of the lungs were increased without reaching statistical significance in all treated test groups 1, 2 and 3. The increased mean absolute and relative weights of the lungs in the main and recovery groups were regarded to be treatment-related as there were changes in a similar direction and magnitude in the main group and control group, the change increased with dose and the absolute weights in the recovery group reached statistical significance in all treated test groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MAIN GROUP
Red discoloration was observed in the lungs and mediastinal lymph nodes in animals of test group 2 and 3, and in tracheobronchial lymph nodes in animals of test group 3. Other findings occurred individually in test group 2. They were considered to be incidental or spontaneous in origin and without any relation to treatment- including one animal with discolored skin, which might have been caused by the test item, but showed no histopathological correlate.

RECOVERY GROUP
Red discoloration was still observed in the lungs of animals of test group 3, and in mediastinal lymph nodes and tracheobronchial lymph nodes in animals of test groups 2 and 3. One finding occurred individually in group 2. It was considered to be incidental or spontaneous in origin and without any relation to treatment.

The red discoloration in lungs and lymph nodes in main and recovery groups was regarded to be treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MAIN GROUP
Treatment-related findings were observed in larynx (level I), lungs, mediastinal lymph nodes, and tracheobronchial lymph nodes.

Larynx, level I: Animals of all test groups showed a minimal focal area with flattened epithelium and loss of cilia (“epithelial alteration”). This was seen with increased incidence in treated animals. The incidence of this finding was regarded to be treatment relatedly increased.

Lungs: The following treatment-related findings were noted in the lungs:
Hyperplasia/hypertrophy of bronchioli, hyperplasia of type II pneumocytes, and intraalveolar cellular debris were noted in animals of test groups 2 (20 mg/m³) and 3 (60 mg/m³). Intraalveolar neutrophils were seen additionally in all test group 3 (60mg/m³) animals. Macrophages containing red particles were seen in clusters aggregates) in alveoli, singly in alveoli or interstitium of alveolar septae and also in the bronchus-associated lymphoid tissue (BALT) in all treated test roups with increasing incidence and/or severity with increasing dose.
Macrophages were also noted in single blood vessels in test groups 2 and 3. Free, unphagocytized particles were seen in the interstitium of the alveolar septae, and also in bronchial epithelial cells in most test group 3 animals and one animal of test group 1.

Mediastinal Lymph Nodes: In the mediastinal lymph nodes of animals of test groups 2 (20 mg/m³) and 3 (60 mg/m³) macrophages were observed that revealed the same red particles as described for the lungs. This finding was regarded to be treatment-related.

Tracheobronchial Lymph Nodes: In the tracheobronchial lymph nodes of animals of test groups 2 (20 mg/m³) and 3 (60 mg/m³) macrophages were observed that revealed the same red particles as described for the lungs. This finding was regarded to be treatment-related.

RECOVERY GROUP
No treatment-related findings were observed in the larynx, level I after the recovery period.

Lungs: Most findings described above for the main group were still noted in animals after the recovery period. Hyperplasia type II pneumocytes was decreased in incidence in test group 3. Hyperplasia/hypertrophy of bronchioli, intraalveolar cellular debris and intraalveolar neutrophils were no longer observed. The macrophages having phagocytized particles were still present after the recovery period: Aggregates were only observed in test groups 2 and 3, and one animal of test group 1 showed an intravascular macrophage. Otherwise, findings were comparable to the main group. Pigment particles deposited in the lungs were still present but with reduced incidence as compared to the main group, no findings were seen in test group 1 recovery animals.

Mediastinal Lymph Nodes: In the mediastinal lymph nodes of animals of test groups 1 (5 mg/m³), 2 (20 mg/m³) and 3 (60 mg/m³) macrophages were still observed that revealed the same red particles as described for
the lungs. This finding was regarded to be treatment-related.

Tracheobronchial Lymph Nodes: In the tracheobronchial lymph nodes of animals of test groups 2 (20 mg/m³) and 3 (60 mg/m³) macrophages were observed that revealed the same red particles as described for the lungs. This finding was 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:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
After the administration period, in BAL of males in test groups 1, 2 and 3 (5, 20 and 60 mg/m³) absolute and relative neutrophil counts were significantly increased. Additionally, in BAL of males in test groups 2 and 3, total cell counts as well as absolute and relative lymphocyte and monocyte counts were increased and only in the high dose group absolute eosinophil cell counts were significantly higher compared to controls (relative lymphocytes in test group 2 not statistically significantly increased). Relative macrophage cell counts were significantly decreased in BAL of males in test groups 1, 2 and 3. These alterations were regarded as treatment related and adverse. 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 groups 2 and 3 (20 and 60 mg/m3) total protein levels as gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities were slightly but significantly increased. Additionally, N-Acetyl-beta-D-glucosaminidase (NAG) activities were significantly increased in test group 3, and GGT activities were already significantly increased in BAL of males in test group 1 (5 mg/m³). These alterations were regarded as treatment related and adverse. In BAL of males of test group 1 (5 mg/m³) total protein levels and LDH and ALP activities were significantly increased but the change was less than 2fold. The same was true for NAG activities in BAL of males in test group 2 (20 mg/m³). Therefore, these alterations were regarded as nonadverse if at all treatment related. After the three-week recovery period, all BAL parameter values were back in the normal range. After the administration period, in BAL of males of test groups 2 and 3 (20 and 60 mg/m³) cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) and osteopontin levels were increased (osteopontin levels in test group 2 not statistically significantly). A slight but significant increase of CINC-1/IL-8 levels was already observed in BAL of males in test group 1 (5 mg/m³). These alterations were regarded as treatment related and adverse.

Details on results:

During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust generation techniques in the concentration range tested. Particle size distribution measurements demonstrated dusts were respirable to rats. The exposure caused no clinical signs of toxicity. It was noted that serval animals showed substance-contaminated fur, or substance like discoloration of the fur. These findings, clearly related to the exposure of the pigment, were non adverse. There was no biologically relevant impairment of body weight development. Regarding clinical pathology, no treatment related, adverse changes of blood parameters were observed after a five-day administration period. In the bronchoalveolar lavage (BAL) of males in test group 2 and 3 (20 and 60 mg/m³) a local inflammation was observed by mainly increased neutrophil cell counts accompanied by increases of lymphocyte and monocyte cell counts as well as total protein levels in BAL and enzyme activity increases (gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in test groups 2 and 3 and N-Acetyl-beta-glucosaminidase (NAG) activities only in test group 3. The cell count increases were reflected by higher CXC cytokine (CINC1-IL-8) and osteopontin levels. In BAL of test group 1 (5 mg/m³) absolute neutrophil cell counts were already 15fold increased and consequently relative neutrophil cell counts as well as CINC-1/IL-8 levels were also significantly higher compared to controls. This was also true for GGT activities. However, the changes in test group 1 were very slight. After a three-week period, all changed BAL parameters recovered completely. Regarding pathology, target organs were larynx (level I), lungs and tracheobronchial and mediastinal lymph nodes.

Main Group:
All animals of test groups 1 (5 mg/m³), 2 (20 mg/m³), and four animals of test group 3 (60 mg/m³) revealed a focal epithelial alteration in the larynx level I at the base of the epiglottis which was also observed in two control animals. As the severity was mostly only minimal and this is a wellknown finding in inhalation studies it was regarded to be treatment related but not an adverse effect (Kaufmann et al., 2009; Renne et al., 2009). In the lungs, hyperplasia/hypertrophy of bronchioli, hyperplasia of type II pneumocytes, intraalveolar cellular debris were noted in animals of test groups 2 (20 mg/m³) and 3 (60 mg/m³). Additionally, intraalveolar neutrophils were seen in all test group 3 (60mg/m³) animals. These findings were assumed to have developed on the background of pigment deposition and the normal macrophage clearance mechanism of the lungs, which might have been overwhelmed. These findings were regarded to be treatment-related and adverse. Furthermore, macrophages containing red particles were seen in clusters (aggregates) in alveoli, singly in alveoli or interstitium of alveolar septae and also in the bronchus-associated lymphoid tissue (BALT) in all treated test groups with increasing incidence and/or severity with increasing dose. Macrophages were also noted in single blood vessels in test groups 2 and 3. These findings were regarded to be treatment-related and representing the normal clearance mechanisms of the lung and therefore not adverse. Pigment deposition without further visible reaction was seen in the interstitium of the alveolar septae, and also in bronchial epithelial cells in most test group 3 animals and one animal of test group 1. This was also regarded to be treatment-related and not adverse. The abovementioned findings in the lung were considered the cause of the increased weights of the lungs in all treated test groups of main groups (without statistical significance) and also of the macroscopically observed discoloration. In test groups 2 and 3, within the mediastinal and tracheobronchial lymph nodes macrophages containing the red particles within their cytoplasm were detected which was the cause for the macroscopically detected discoloration of the lymph nodes. This finding was regarded to be treatment-related but not adverse as it is a normal route of lung clearance, and no additional findings were observed.

Recovery group
No treatment-related increase of epithelial alteration in the larynx was noted after the recovery period. In the lungs, hyperplasia type II pneumocytes was decreased in incidence and severity and hyperplasia/hypertrophy of bronchiole, intraalveolar cellular debris and intraalveolar neutrophils were no longer seen. The hyperplasia of type II pneumocytes in conjunction with deposition and clearance of red particles in the lungs was regarded to be treatment-related and adverse. Macrophage aggregates, single macrophages in alveoli or interstitium of alveolar septae or in single blood vessels and also in the bronchus-associated lymphoid tissue (BALT) were still seen in all treated test groups. Aggregates were only observed in test groups 2 and 3, and one animal of test group 1 showed an intravascular macrophage. Otherwise, findings were comparable to the main group. These findings were regarded to be treatment-related and representing the normal clearance mechanisms of the lung and therefore not adverse. Particle deposition without further visible reaction in the interstitium of the alveolar septae was still observed in test group 3, and also in bronchial epithelial cells in test group 2 and regarded to be treatment-related and not adverse. The abovementioned findings in the lung were considered the cause of the increased weights of the lungs in all treated test groups of recovery groups (with statistical significance only in absolute weights in the recovery group) and also of the macroscopically observed discoloration. In all treated groups within the mediastinal and tracheobronchial lymph nodes macrophages containing the red particles within their cytoplasm were still detected which was the cause for treatment-related but not adverse as it is a normal route of lung clearance, and no additional findings were observed.

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.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

>= 60 mg/m³ air

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

5 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: parameters in branchoalveolar lavage fluid

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air

System:

other: respiratory system and lymph nodes

Organ:

larynx

lungs

lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

NOAEC(systemic) > 60 mg/m³, LOAEC(local) = 5 mg/m³ (reversible)

Executive summary:

In a reliable GLP-conform study similar to OECD TG 412 (5 days instead of 28 days of exposure) 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 was determined. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of 4,11-dichloro-5,12- dihydroquino[2,3-b]acridine-7,14-dione 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 nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 5, 20, and 60 mg/m3 (low, mid, and high concentration) for 6 hours per day and 5 days. 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, gross pathology and all histopathological changes. During the exposure period the target concentrations were reached. The particle size resulted in MMADs between 0.76 and 1.07 μm with GSDs between 2.73 and 3.34. The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 82.4 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. The following substance-relative adverse findings were observed:

 

During the exposure period, clinical signs of toxicity limited to discoloration of the fur and contamination with the test item, which was a result to the pigment and not an adverse finding. There was no substance-related impairment of body weight development.

 

Test group 3 (main group, 60 mg/m³)

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


• Increased absolute eosinophil cell counts in BAL


• Decreased relative macrophage counts in BAL


• Increased total protein levels as well as gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and N-acetyl-beta-glucosaminidase(NAG) activities in BAL


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


• Hyperplasia/hypertrophy of bronchioli, hyperplasia of type II pneumocytes, intraalveolar cellular debris, and infiltration of neutrophils in alveoli associated with deposition and clearance of red particles in the lungs

 

Test group 3 (recovery group, 60 mg/m³)

• Hyperplasia of type II pneumocytes, associated with deposition and clearance of red particles in the lungs

 

Test group 2 (main group, 20 mg/m³)

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


• Decreased relative macrophage counts in BAL


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


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


• Hypertrophy/ hyperplasia of terminal bronchioles, hyperplasia of type II pneumocytes, intraalveolar cellular debris associated with deposition and clearance of red particles in the lungs

 

Test group 2 (recovery group, 20 mg/m3)

• Hyperplasia of type II pneumocytes, associated with deposition and clearance of red particles in the lungs

 

Test group 1 (main group, 5 mg/m³)

• Increased absolute and relative neutrophil cell counts in BAL


• Decreased relative macrophage counts in BAL


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


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

 

Test group 1 (recovery group, 5 mg/m³)

• No adverse effects

 

Inhalation exposure of rats up to 60 mg/m³ 4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione on 5 consecutive days did not cause any systemic effects. However, changes of several parameters in BAL were detected in all test groups in a concentration-related manner. In the lungs, hyperplasia/hypertrophy of bronchioli, hyperplasia of type II pneumocytes, intra-alveolar cellular debris were observed in animals exposed to 20 and 60 mg/m³. At the high concentration (60 mg/m³), intraalveolar neutrophils were observed in addition. After the post-exposure period of 3 weeks, the effects in BAL resolved completely in all test groups. At the mid (20 mg/m³) and high concentrations (60 mg/m³) hyperplasia of type II pneumocytes was still observed which was accompanied by slightly increased relative lung weights. Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for systemic effect was above the highest tested concentration of 60 mg/m³. A NOAEC for local effects could not be determined, although 5 mg/m³ could be considered as the low observed adverse effect concentration (LOAEC). The local effects were principally reversible as all BAL parameters returned to the control level and the histological finding mostly regressed after 3 weeks post-exposure observation period.