3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

Endpoint summary

Description of key information

Oral: 

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study, according to OECD 422, GLP, rat, NOAEL for general systemic toxicity: 1000 mg/kg bw/d (no mortality or signs of toxicity)

 

Inhalation:

Short-term Inhalation Study, similar to OECD 412, GLP, rat, 5-day treatment with 21 days recovery, NOAEC for systemic effects: 60 mg/m³ air, NOAEC for local effects: 20 mg/m³ air

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2011 - June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation: not specified
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 9th, 2011 To: October 6th, 2011

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

-Applied as a suspension in water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- according to GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina

Statistics:

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

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- All male animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study, i.e. on study days 33 and 34. Also all female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal of test group 1 (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- In males of test groups 1 and 2 (100 and 300 mg/kg bw/d), creatinine values were lower compared to controls, but the means were not dose-dependently decreased. Therefore, this alteration was regarded as incidental and not treatment-related.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 9 in male animals and increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no dose-response relationship was observed, the findings were assessed as being incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- The weight increase in absolute and relative spleen weight in females and in relative spleen weight in males of test group 2 (300 mg/kg bw/d) was regarded to be incidental due to a missing dose-response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw/d).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 2 males and 7 females of test group 3 (1000 mg/kg bw/d) revealed a yellow discoloration of the glandular stomach contents. 3 females of test group 3 (1000 mg/kg bw/d) showed the same discoloration of the contents of the jejunum.
- One female animal of test group 1 (100 mg/kg bw/d) revealed a yellow discoloration of the lung (regarded to be test substance that was aspired subsequently to the gavage procedure or due to a gavage error into the trachea) and the mediastinal lymph nodes (regarded to be the physiologic clearing route of the lung).

These discolorations were caused by the test substance but were not regarded to be a treatment-related adverse finding.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Remarks on result:

other: no adverse effects observed up to and including the limit dose

Critical effects observed:

no

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

According to OECD TG 422, GLP, Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Aug 2020 - 23 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

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

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation (means):
- Housing: The rats were housed together (up to 5 animals per cage)
- Diet: Mouse/rat laboratory diet; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.42 - <= 0.67 µm

Remarks on MMAD:

Please, see Table 1 in section "Any other information on materials and methods incl. tables"

Details on inhalation exposure:

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) was used for generating the dust. The concentration 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.

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel 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 head-nose exposure technique was preferably selected for this aerosol/dust/ 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 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 two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

The air flows were constantly maintained in the desired range.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please, see Table 2 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

Dose / conc.:

5 mg/m³ air

Remarks:

Measured concentration: 5.1 +/- 0.2 mg/m³

Dose / conc.:

20 mg/m³ air

Remarks:

Measured concentration: 19.3 +/- 0.4 mg/m³

Dose / conc.:

60 mg/m³ air

Remarks:

Measured concentration: 60.6 +/- 2.0 mg/m³

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Observations and examinations performed and frequency:

MORTALITY: The animals were examined for evident signs of toxicity or mortality 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: The clinical condition of the test animals was recorded 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: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).
ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA . Rat haptoglobin was measured with an ELISA.

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. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer. Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA , Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA, Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA , Rodent osteopontin measured with an ELISA .

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: 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 and eyelids, 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. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy. Tissues and organs to be examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).

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
- 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: 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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period and the post-exposure observation period the animals showed no clinical signs and findings different from normal, as well as the animals of the control group and low concentration (5 mg/m³) during the exposure period. At mid (20 mg/m³) and high (60 mg/m³) the animals showed substance contaminated fur after daily exposure.

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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No substance-related changes of food consumption were observed during the whole study period.

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 the end of the administration period, in males of test group 3 (60 mg/m3) absolute and relative neutrophil counts were significantly increased. However, the values were marginally above the historical control range (absolute neutrophils) or within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 10.0-17.9 %). Therefore, these alterations were regarded as incidental and not treatment related

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were 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, non-treatment-related

Description (incidence and severity):

Main groups:
All mean absolute weight parameters did not show significant differences when compared to the control group.
When compared to control group (=100%), the mean relative weights of the adrenals were significantly increased in group 2 (20 mg/m³): 118 %; but not in the other dose groups. Due to a missing dose-response relationship the increase was regarded as incidental.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Recovery groups:
All mean absolute weight parameters did not show significant differences when compared to the control group.
Due to a missing dose-response relationship the decrease in relative thymus weight (73 %, 20 mg/m³) was regarded as incidental. The increased relative thyroid glands weight (126 %, 60 mg/m³) was within historical control values and therefore as well considered as 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, treatment-related

Description (incidence and severity):

All animals of test group 3 (60 mg/m³) of the main and recovery group revealed a yellow discoloration in the lungs, mediastinal and tracheobronchial lymph nodes. This 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.

Neuropathological findings:

not examined

Description (incidence and severity):

Main group:
In the lungs animals of test group 2 and 3 (20 and 60 mg/m³) showed increased numbers of histiocytes (histiocytosis) within the alveoli which revealed yellow particles within their cytoplasm. In test group 1 (5 mg/m³) the histiocytes were not increased in number but also contained yellow particles inside.
All animals of test group 3 (60 mg/m³) had in addition to the increased numbers of histiocytes also minimal infiltration with neutrophils and a hypertrophy/hyperplasia of the epithelium of the terminal bronchioles.
In some animals of all treated groups occasionally single macrophages with yellow particles in their cytoplasm were observed intravascularly. All these findings were regarded to be treatment-related.
At the base of the epiglottis of all animals of test group 3 (60 mg/m³) a minimal focal area with flattened epithelium and loss of cilia was seen in three of the five animals. This finding was regarded to be treatment related.
The tracheobronchial lymph node was chosen exemplarily, similar findings were observed in the mediastinal lymph nodes. Within the lymph node of animals of all test groups histiocytes were observed containing the same yellow particles inside their cytoplasm as observed in the lungs. This 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.

Recovery group:
In the lung animals of test group 3 (60 mg/m³) showed increased numbers of histiocytes (histiocytosis) which revealed yellow particles within their cytoplasm. In test group 1 and 2 (5 and 20 mg/m³) the histiocytes were not increased in number but also contained yellow particles inside. Some animals of all treated groups showed single macrophages with yellow particles in
the bronchus-associated lymphoid tissue (BALT).
In some animals of test groups 2 and 3 (20 and 60 mg/m³) occasionally single macrophages with yellow particles in their cytoplasm were observed intravascularly. All these findings were regarded to be treatment-related.
Some animals of all treated groups still had macrophages with yellow particles within the draining lymph nodes of the lung. In test group 3 (60 mg/m³) these macrophages had formed small aggregates in addition. 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 specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL):
At the end of the administration period, in males of test group 3 (60 mg/m3) total cell counts in BAL were significantly increased. This increase was mainly due to increased absolute neutrophil counts followed by increases of absolute lymphocyte and monocyte counts in BAL (not statistically significant). Additionally, in the BAL of these individuals, higher levels of bronchial epithelial cells were found (not statistically significant). These changes were regarded as treatment related and adverse.
After three weeks of recovery, in male rats of test group 1 (5 mg/m3) absolute lymphocyte counts in BAL were significantly increased, and in males of test group 2 (20 mg/m3) relative neutrophil counts in BAL were significantly higher compared to study controls. Additionally, in males of test groups 2 and 3 (20 and 60 mg/m3) relative macrophage counts were significantly decreased. However, all mentioned changes in this paragraph were not dose dependent and therefore, they were regarded as incidental and not treatment related.
At the end of the administration period, in males of test group 3 (60 mg/m3) total protein levels in BAL γ-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities in BAL were significantly increased. Alkaline phosphatase (ALP) and N-Acetyl-β-D-glucosaminidase (NAG) activities in BAL were also significantly increased in this test group, but ALP was only marginal higher compared to controls, and the NAG increase was below 2fold. Therefore, total protein level as well as GGT, LDH and ALP activity increases in BAL of test group 3 were regarded as treatment-related and adverse, whereas the NAG activity increase was regarded as maybe treatment-related but non-adverse.
After the 3-week recovery period, no significant changes of the mentioned parameters occurred.

Antigens in BAL:
After the administration period, in males of test group 3 (60 mg/m3) monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL were significantly increased. These alterations were regarded as treatment related and adverse.
After the recovery period, in males of test group 2 (20 mg/m3) CINC-1/IL-8 levels in BAL were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

>= 60 mg/m³ air

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

20 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Please see section "Any other information on results incl. tables"

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

The following local effects were observed after treatment with 60 mg/m^3:

• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial epithelial cells in BAL


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


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


• Increased histiocytes with intracytoplasmic yellow particles,


• hypertrophy/ hyperplasia of terminal bronchioles,


• infiltration of neutrophils

Data from lavage an histology were consistent with the above mentioned effects. All effects were reversible after a 3-week recovery period.

 

Table 4: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
Total Cells	1.2	1.4	3.7**	1.0	0.8	0.8
Eosinophils	0.1	1.5	0.2	+	+	+
Lymphocytes	1.9	1.8	7.5	7.1*	5.7	2.8
Macrophages	1.2	1.3	2.7	1.0	0.8	0.8
Neutrophils	0.7	2.6	79.0	1.0	3.3	1.8
Monocytes	0.0	3.4	121.6	1.0	1.0	1.8
Epithelial cells	2.1	29.2	50.3	+	+	+

One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

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

 

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

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
Total Protein	1.2	1.3	2.3**	1.3	1.2	1.2
GGT	1.3	1.4	3.7**	1.0	0.9	0.9
LDH	1.7	1.8	5.0**	1.6	1.2	1.3
ALP	1.1	1.0	2.3**	1.1	1.1	1.0
NAG	1.0	1.4	1.8*	1.3	1.0	1.2

GGT = gamma-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = N-Acetyl-beta-D-glucosaminidase, One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

 

Table 6: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
MCP-1	1.3	1.0	7.5**	0.8	1.0	1.2
CINC-1/IL-8	1.0	0.8	2.3**	0.8	1.6**	1.2
M-CSF	1.0	1.0	1.3	1.0	1.0	1.0
Osteopontin	0.3	0.7	2.4	0.3	1.2	1.2

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

One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

 

Table 7: Relative changes of relative organ weights in main group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Adrenal glands	98%	118%*	117%

*p ≤ 0.05; **p ≤ 0.01

 

Table 8: Relative changes of relative organ weights in recovery group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Thymus	82%	73%*	90%
Thyroid glands	111%	96%	126%*

*p ≤ 0.05; **p ≤ 0.01

 

Table 9: Gross lesions observed in main group animals (F1)

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs ·        Discoloration	0	0	5	5
Mediastinal lymph node ·        Discoloration	0	0	0	5
Tracheobronchial lymph node ·        Discoloration	0	0	0	5

 

Table 10: Gross lesions observed in recovery group animals (R1)

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs ·        Discoloration	0	0	0	5
Mediastinal lymph node ·        Discoloration	0	0	0	5
Tracheobronchial lymph node ·        Discoloration	0	0	0	5

 

Table 11: Incidence and severity of histological findings in the lung of main group animals (F1)

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Histiocytosis, alveolar with particles	0	0	5	5
·        Grade 1			5
·        Grade 2				5
Particles within histiocytes	0	5	0	0
Infiltration neutrophils (m)f	0	0	0	5
·        Grade 1				5
Hypertrophy/hyperplasia term. bronchi	0	0	0	5
·        Grade 1				3
·        Grade 2				2
Macrophages w. particles iv	0	2	4	3

All findings that were not graded were recorded as present

 

Table 12: Incidence and severity of histological findings in the larynx of main group animals (F1)

Larynx (level I)	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Epithelial alteration, (multi)focal	0	0	0	3
·        Grade 1				3

 

Table 13: Incidence and severity of histological findings in the tracheobronchial lymph nodes of main group animals (F1)

Tracheobronchial lymph node	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	4	5	5
Macrophages with particles	0	1	3	5
·        Grade 1		1	3	2
·        Grade 2				3

 

Table 14: Incidence and severity of histological findings in the lungs of recovery group animals (R1)

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Histiocytosis, alveolar w. particles	0	0	0	5
·        Grade 2				5
Particles within histiocytes	0	5	5	0
BALT: macrophages with particles	0	3	5	5
·        Grade 1		3	5	5
Macrophages w. particles iv	0	0	1	3

All findings that were not graded were recorded as present

 

Table 15: Incidence and severity of histological findings in the tracheobronchial lymph nodes of recovery group animals (R1)

Tracheobronchial lymph node	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles	0	3	5	1
·        Grade 1		3	4	1
·        Grade 2			1
Macrophage aggregates with particles	0	0	0	4
·        Grade 1				1
·        Grade 2				3

Conclusions:

Inhalation exposure of rats to 60 mg/m³ of the test substance on 5 consecutive days caused increased lymphocytes, neutrophils, monocyte counts and epithelial cells in bronchoalveolar lavage. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as increased histiocytes with intracytoplasmic yellow particles, hypertrophy/hyperplasia of terminal bronchioles. All effects were reversible within 3 weeks exposure-free recovery period.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 20 mg/m³ for increased histiocytes with intracytoplasmic yellow particles, hypertrophy/ hyperplasia of terminal bronchioles. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ is the lung.

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 subatance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.
For this purpose groups of 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 and all histopathological changes.

The particle size resulted in MMADs between 0.42 and 0.67 μm with GSDs between 2.50 and 3.59. The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 90 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

 

The following substance-related adverse findings were observed:

 

Test group 3 (60 mg/m³)
• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial
epithelial cells in BAL
• Increased total protein levels as well as γ-glutamyl transferase (GGT), lactate
dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL
• Increased monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced
neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL
• Increased histiocytes with intracytoplasmic yellow particles, hypertrophy/ hyperplasia of
terminal bronchioles, and infiltration of neutrophils

 

Test group 2 (20 mg/m³) and Test group 1 (5 mg/m³)
No adverse effects were observed.

 

Recovery group
No adverse effects were observed regarding pathology findings.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

60 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Similar to OECD TG 412, GLP, Klimisch 1

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Aug 2020 - 23 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

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

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation (means):
- Housing: The rats were housed together (up to 5 animals per cage)
- Diet: Mouse/rat laboratory diet; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.42 - <= 0.67 µm

Remarks on MMAD:

Please, see Table 1 in section "Any other information on materials and methods incl. tables"

Details on inhalation exposure:

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) was used for generating the dust. The concentration 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.

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel 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 head-nose exposure technique was preferably selected for this aerosol/dust/ 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 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 two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

The air flows were constantly maintained in the desired range.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please, see Table 2 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

Dose / conc.:

5 mg/m³ air

Remarks:

Measured concentration: 5.1 +/- 0.2 mg/m³

Dose / conc.:

20 mg/m³ air

Remarks:

Measured concentration: 19.3 +/- 0.4 mg/m³

Dose / conc.:

60 mg/m³ air

Remarks:

Measured concentration: 60.6 +/- 2.0 mg/m³

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Observations and examinations performed and frequency:

MORTALITY: The animals were examined for evident signs of toxicity or mortality 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: The clinical condition of the test animals was recorded 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: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).
ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA . Rat haptoglobin was measured with an ELISA.

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. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer. Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA , Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA, Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA , Rodent osteopontin measured with an ELISA .

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: 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 and eyelids, 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. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy. Tissues and organs to be examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).

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
- 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: 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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period and the post-exposure observation period the animals showed no clinical signs and findings different from normal, as well as the animals of the control group and low concentration (5 mg/m³) during the exposure period. At mid (20 mg/m³) and high (60 mg/m³) the animals showed substance contaminated fur after daily exposure.

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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No substance-related changes of food consumption were observed during the whole study period.

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 the end of the administration period, in males of test group 3 (60 mg/m3) absolute and relative neutrophil counts were significantly increased. However, the values were marginally above the historical control range (absolute neutrophils) or within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 10.0-17.9 %). Therefore, these alterations were regarded as incidental and not treatment related

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were 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, non-treatment-related

Description (incidence and severity):

Main groups:
All mean absolute weight parameters did not show significant differences when compared to the control group.
When compared to control group (=100%), the mean relative weights of the adrenals were significantly increased in group 2 (20 mg/m³): 118 %; but not in the other dose groups. Due to a missing dose-response relationship the increase was regarded as incidental.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Recovery groups:
All mean absolute weight parameters did not show significant differences when compared to the control group.
Due to a missing dose-response relationship the decrease in relative thymus weight (73 %, 20 mg/m³) was regarded as incidental. The increased relative thyroid glands weight (126 %, 60 mg/m³) was within historical control values and therefore as well considered as 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, treatment-related

Description (incidence and severity):

All animals of test group 3 (60 mg/m³) of the main and recovery group revealed a yellow discoloration in the lungs, mediastinal and tracheobronchial lymph nodes. This 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.

Neuropathological findings:

not examined

Description (incidence and severity):

Main group:
In the lungs animals of test group 2 and 3 (20 and 60 mg/m³) showed increased numbers of histiocytes (histiocytosis) within the alveoli which revealed yellow particles within their cytoplasm. In test group 1 (5 mg/m³) the histiocytes were not increased in number but also contained yellow particles inside.
All animals of test group 3 (60 mg/m³) had in addition to the increased numbers of histiocytes also minimal infiltration with neutrophils and a hypertrophy/hyperplasia of the epithelium of the terminal bronchioles.
In some animals of all treated groups occasionally single macrophages with yellow particles in their cytoplasm were observed intravascularly. All these findings were regarded to be treatment-related.
At the base of the epiglottis of all animals of test group 3 (60 mg/m³) a minimal focal area with flattened epithelium and loss of cilia was seen in three of the five animals. This finding was regarded to be treatment related.
The tracheobronchial lymph node was chosen exemplarily, similar findings were observed in the mediastinal lymph nodes. Within the lymph node of animals of all test groups histiocytes were observed containing the same yellow particles inside their cytoplasm as observed in the lungs. This 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.

Recovery group:
In the lung animals of test group 3 (60 mg/m³) showed increased numbers of histiocytes (histiocytosis) which revealed yellow particles within their cytoplasm. In test group 1 and 2 (5 and 20 mg/m³) the histiocytes were not increased in number but also contained yellow particles inside. Some animals of all treated groups showed single macrophages with yellow particles in
the bronchus-associated lymphoid tissue (BALT).
In some animals of test groups 2 and 3 (20 and 60 mg/m³) occasionally single macrophages with yellow particles in their cytoplasm were observed intravascularly. All these findings were regarded to be treatment-related.
Some animals of all treated groups still had macrophages with yellow particles within the draining lymph nodes of the lung. In test group 3 (60 mg/m³) these macrophages had formed small aggregates in addition. 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 specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL):
At the end of the administration period, in males of test group 3 (60 mg/m3) total cell counts in BAL were significantly increased. This increase was mainly due to increased absolute neutrophil counts followed by increases of absolute lymphocyte and monocyte counts in BAL (not statistically significant). Additionally, in the BAL of these individuals, higher levels of bronchial epithelial cells were found (not statistically significant). These changes were regarded as treatment related and adverse.
After three weeks of recovery, in male rats of test group 1 (5 mg/m3) absolute lymphocyte counts in BAL were significantly increased, and in males of test group 2 (20 mg/m3) relative neutrophil counts in BAL were significantly higher compared to study controls. Additionally, in males of test groups 2 and 3 (20 and 60 mg/m3) relative macrophage counts were significantly decreased. However, all mentioned changes in this paragraph were not dose dependent and therefore, they were regarded as incidental and not treatment related.
At the end of the administration period, in males of test group 3 (60 mg/m3) total protein levels in BAL γ-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities in BAL were significantly increased. Alkaline phosphatase (ALP) and N-Acetyl-β-D-glucosaminidase (NAG) activities in BAL were also significantly increased in this test group, but ALP was only marginal higher compared to controls, and the NAG increase was below 2fold. Therefore, total protein level as well as GGT, LDH and ALP activity increases in BAL of test group 3 were regarded as treatment-related and adverse, whereas the NAG activity increase was regarded as maybe treatment-related but non-adverse.
After the 3-week recovery period, no significant changes of the mentioned parameters occurred.

Antigens in BAL:
After the administration period, in males of test group 3 (60 mg/m3) monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL were significantly increased. These alterations were regarded as treatment related and adverse.
After the recovery period, in males of test group 2 (20 mg/m3) CINC-1/IL-8 levels in BAL were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

>= 60 mg/m³ air

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

20 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Please see section "Any other information on results incl. tables"

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

The following local effects were observed after treatment with 60 mg/m^3:

• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial epithelial cells in BAL


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


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


• Increased histiocytes with intracytoplasmic yellow particles,


• hypertrophy/ hyperplasia of terminal bronchioles,


• infiltration of neutrophils

Data from lavage an histology were consistent with the above mentioned effects. All effects were reversible after a 3-week recovery period.

 

Table 4: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
Total Cells	1.2	1.4	3.7**	1.0	0.8	0.8
Eosinophils	0.1	1.5	0.2	+	+	+
Lymphocytes	1.9	1.8	7.5	7.1*	5.7	2.8
Macrophages	1.2	1.3	2.7	1.0	0.8	0.8
Neutrophils	0.7	2.6	79.0	1.0	3.3	1.8
Monocytes	0.0	3.4	121.6	1.0	1.0	1.8
Epithelial cells	2.1	29.2	50.3	+	+	+

One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

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

 

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

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
Total Protein	1.2	1.3	2.3**	1.3	1.2	1.2
GGT	1.3	1.4	3.7**	1.0	0.9	0.9
LDH	1.7	1.8	5.0**	1.6	1.2	1.3
ALP	1.1	1.0	2.3**	1.1	1.1	1.0
NAG	1.0	1.4	1.8*	1.3	1.0	1.2

GGT = gamma-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = N-Acetyl-beta-D-glucosaminidase, One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

 

Table 6: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (2 days after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³	Gr. 1 5 mg/m³	Gr. 2 20 mg/m³	Gr. 3 60 mg/m³
MCP-1	1.3	1.0	7.5**	0.8	1.0	1.2
CINC-1/IL-8	1.0	0.8	2.3**	0.8	1.6**	1.2
M-CSF	1.0	1.0	1.3	1.0	1.0	1.0
Osteopontin	0.3	0.7	2.4	0.3	1.2	1.2

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

One-sided Wilcoxon-test: * : p ≤ 0.05; ** : p ≤ 0.01

 

Table 7: Relative changes of relative organ weights in main group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Adrenal glands	98%	118%*	117%

*p ≤ 0.05; **p ≤ 0.01

 

Table 8: Relative changes of relative organ weights in recovery group animals

	Male animals
Test group (mg/m³)	1 (5)	2 (20)	3 (60)
Thymus	82%	73%*	90%
Thyroid glands	111%	96%	126%*

*p ≤ 0.05; **p ≤ 0.01

 

Table 9: Gross lesions observed in main group animals (F1)

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs ·        Discoloration	0	0	5	5
Mediastinal lymph node ·        Discoloration	0	0	0	5
Tracheobronchial lymph node ·        Discoloration	0	0	0	5

 

Table 10: Gross lesions observed in recovery group animals (R1)

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs ·        Discoloration	0	0	0	5
Mediastinal lymph node ·        Discoloration	0	0	0	5
Tracheobronchial lymph node ·        Discoloration	0	0	0	5

 

Table 11: Incidence and severity of histological findings in the lung of main group animals (F1)

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Histiocytosis, alveolar with particles	0	0	5	5
·        Grade 1			5
·        Grade 2				5
Particles within histiocytes	0	5	0	0
Infiltration neutrophils (m)f	0	0	0	5
·        Grade 1				5
Hypertrophy/hyperplasia term. bronchi	0	0	0	5
·        Grade 1				3
·        Grade 2				2
Macrophages w. particles iv	0	2	4	3

All findings that were not graded were recorded as present

 

Table 12: Incidence and severity of histological findings in the larynx of main group animals (F1)

Larynx (level I)	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Epithelial alteration, (multi)focal	0	0	0	3
·        Grade 1				3

 

Table 13: Incidence and severity of histological findings in the tracheobronchial lymph nodes of main group animals (F1)

Tracheobronchial lymph node	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	4	5	5
Macrophages with particles	0	1	3	5
·        Grade 1		1	3	2
·        Grade 2				3

 

Table 14: Incidence and severity of histological findings in the lungs of recovery group animals (R1)

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Histiocytosis, alveolar w. particles	0	0	0	5
·        Grade 2				5
Particles within histiocytes	0	5	5	0
BALT: macrophages with particles	0	3	5	5
·        Grade 1		3	5	5
Macrophages w. particles iv	0	0	1	3

All findings that were not graded were recorded as present

 

Table 15: Incidence and severity of histological findings in the tracheobronchial lymph nodes of recovery group animals (R1)

Tracheobronchial lymph node	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles	0	3	5	1
·        Grade 1		3	4	1
·        Grade 2			1
Macrophage aggregates with particles	0	0	0	4
·        Grade 1				1
·        Grade 2				3

Conclusions:

Inhalation exposure of rats to 60 mg/m³ of the test substance on 5 consecutive days caused increased lymphocytes, neutrophils, monocyte counts and epithelial cells in bronchoalveolar lavage. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as increased histiocytes with intracytoplasmic yellow particles, hypertrophy/hyperplasia of terminal bronchioles. All effects were reversible within 3 weeks exposure-free recovery period.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 20 mg/m³ for increased histiocytes with intracytoplasmic yellow particles, hypertrophy/ hyperplasia of terminal bronchioles. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ is the lung.

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 subatance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.
For this purpose groups of 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 and all histopathological changes.

The particle size resulted in MMADs between 0.42 and 0.67 μm with GSDs between 2.50 and 3.59. The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 90 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

 

The following substance-related adverse findings were observed:

 

Test group 3 (60 mg/m³)
• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial
epithelial cells in BAL
• Increased total protein levels as well as γ-glutamyl transferase (GGT), lactate
dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL
• Increased monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced
neutrophil chemoattractant-1 (CINC-1/IL-8) levels in BAL
• Increased histiocytes with intracytoplasmic yellow particles, hypertrophy/ hyperplasia of
terminal bronchioles, and infiltration of neutrophils

 

Test group 2 (20 mg/m³) and Test group 1 (5 mg/m³)
No adverse effects were observed.

 

Recovery group
No adverse effects were observed regarding pathology findings.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

20 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Similar to OECD TG 412, GLP, Klimisch 1

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

In the course of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422, the test item was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (drinking water served as vehicle), 100 mg/kg bw/d, 300 mg/kg bw/d and 1000 mg/kg bw/d. The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. A detailed clinical observation was performed in all animals. Body weights and food consumption were determined in F0 animals. Clinico-chemical and hematological examinations as well as urinalyses and FOB were performed in all animals towards the end of the administration period. All animals were assessed by gross pathology; weights of selected organs were recorded and a histopathological examination was performed. Clinical examinations, reproductive performance, clinical pathology, histopathology and gross pathology did not reveal any findings in treated animals. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes. 

 

Inhalation:

A 5-day inhalation study similar to OECD 412 in GLP-compliance was performed with the test substance.

Male Wistar rats were exposed to dust-aerosols at concentrations of 5, 20, 60 mg/m³ of the test substance for 5 consecutive days. 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 0.42 and 0.67 μm with GSDs between 2.50 and 3.59. The calculated mass fractions of particles below 3 μm aerodynamic size was greater than 90 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. No systemic effects were observed, therefore, the systemic NOAEC was set above 60 mg/m³. The local NOAEC was set at 20 mg/m³ based on the following findings:

• Increased total cell counts, absolute neutrophil, lymphocyte, monocyte and bronchial epithelial cells in BAL


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


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


• Increased histiocytes with intracytoplasmic yellow particles,


• Hypertrophy/ hyperplasia of terminal bronchioles,


• Infiltration of neutrophiles.

The target oran is the lung.

Data from lavage and histology were consistent with the above mentioned effects. All effects were reversible after a 3-week recovery period.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.