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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-08-04 to 2021-02-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2020-05-11 to 2020-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted as a dose range finding study to investigate the toxicity of Pigment 4 using clinical observation, gross pathology, histopathology and bronchoalveolar lavage fluid (BALF) analysis. However, the food consumption was not recorded.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
2018-06-25
Deviations:
yes
Remarks:
Study conducted as range-finding study for main study.
GLP compliance:
no
Remarks:
The investigations in DRF B were conducted under non-GLP conditions because of the dose-range finding nature of the experiment. However, the experimental procedures followed the GLP rules (e.g. use of SOPs and documentation/archiving).
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. the specified Wistar strain is preferred over the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

*Reference:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 8 weeks old
- Weight at study initiation: approx. 247 g (males) and approx. 170 g (females)
- Housing: animals were housed in Makrolon® (polycarbonate) cages type III with absorbing softwood ('ssniff BK 8-15') bedding material.
- Diet (ad libitum): commercial chow in pellet form (ssniff "V1534", supplier: ssniff Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: approx. one week the animals were allowed to adjust and become acclimatized to the Fraunhofer ITEM environment. During the 2-3 weeks prior to exposure start, all rats were trained to the 6-hour restraint in nose-only tubes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.42 - <= 2.17 µm
Remarks on MMAD:
MMAD / GSD: please refer to table 1 ('Any other information on materials and methods incl. tables').
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: acrylic tubes with a flexible stopper
- System of generating particulates/aerosols: the test item was aerosolized using a dry dispersion system optimized for powdered substances and operated with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. The signal of an aerosol photometer was used to control the feed rate of the dispersion system in order to keep the aerosol concentration in the inhalation unit constant. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test substance under computerized control, i.e. with a feedback loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gave a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.

- Temperature, humidity, pressure in air chamber: parameters were recorded by 20-minute means. The limits were set at 22 ± 2 °C for temperature and 55 ± 15 % for relative humidity.
- Air flow rate: approximately 1 L/min
- Method of particle size determination: the MMAD was determined twice (once before exposure start and once during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor)).
- Treatment of exhaust air: exhaled air was drawn off immediately by a cylinder surrounding the aerosol delivering cylinder.

TEST ATMOSPHERE
- Brief description of analytical method used: filter samples of the aerosols were taken once per day to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close or exactly the target concentrations.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See above in field "details on inhalation exposure".

The target aerosol concentrations of 1.5, 6 and 24 mg/m³ high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix were achieved satisfactorily, i.e. to 100, 96 and 100 %.
Duration of treatment / exposure:
14 days
Frequency of treatment:
6 hours per day, 5 days/week
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.18 mg/m³
Dose / conc.:
6 mg/m³ air (analytical)
Remarks:
SD: ± 0.74 mg/m³
Dose / conc.:
24 mg/m³ air (analytical)
Remarks:
SD: ± 0.85 mg/m³
No. of animals per sex per dose:
5 males/females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a dosing scheme of 1.5, 6 and 24mg/m³ was chosen based on the findings from the intratracheal dose-range finding study (DRF A; study no. 02 N 20 502).
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs): daily, twice during exposure period (with the exception of weekends and public holidays: once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: on day -1 before treatment and twice a week during the exposure period for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 day after last exposure
- Dose groups that were examined: control and all dose groups
- Number of animals: 5 animals/group
- Parameters examined: total cell count, differential cell count (macrophages, PMN, lymphocytes, lactate dehydrogenase (LDH), β-glucuronidase, total protein)

LUNG BURDEN: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
One day after the exposure period, all animals were anesthetized and exsanguinated. All animals were subjected to a complete necropsy.

ORGAN WEIGHTS: Yes
The lung and the lower half of the trachea were weighed (= lung wet weight).

HISTOPATHOLOGY: Yes
histopathology was performed in all animals of the control and pigment 4 high dose group after end of exposure for the following organ tissues: lung lobes, including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of group 1 and 4.
Lungs were divided in two halves for BAL analysis and histopathology. Left lobe and right lobes were taken for histopathology and BALF analysis, respectively. The lung lobe used for histopathology was inflated under a pressure of about 20 cm water with formalin and fixed by immersion for a minimum of 2 hours. Lung lobe was fixed in buffered formalin (10 %) for up to one day, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). In addition, masson trichrome staining was used for detection of connective tissue production within the lung. Bones were decalcified prior to embedding.
All tissues listed in OECD Guideline no. 413 and OPPTS 870.3465 were preserved for histopathology.
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test. The statistical evaluation of the histopathological findings were done with the two-tailed Fisher test by the PROVANTIS system.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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):
Means of terminal body, absolute and relative lung weights did not show any statistically significant changes in the pigment 4 low-dose group as compared to clean air controls. For the pigment mid- and high-dose groups the absolute and relative lung wet weights (5.88 and 6.64 g/kg bw) are statistically significantly higher in both sexes compared to the controls (3.98 g/kg bw; p<0.01).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon necropsy, test item- or dose-related macroscopical findings were observed in form of enlarged lung associated lymph nodes (LALN) in the mid and high dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the laryngopharynx, the larynx, the nasal cavity, the nasopharynx and the trachea, no pathologic findings were observed.
In the lung, exposure-related findings represent multifocal slight alveolar accumulation of particle-laden macrophages in all males and females, multifocal alveolar infiltration of granulocytic cells in all males (4/5 slight and 1/5 moderate) and all females (all slight) as well as multifocal alveolar granular material in all males (3/5 slight and 2/5 moderate) and all females (all moderate). This granular material was free within the alveoli and was interpreted to be at least partially cellular debris. The granular material was mixed sometimes with particles. Further, the amount of alveolar macrophages was additionally increased (alveolar histiocytosis) in all males (1/5 very slight and 4/5 slight) and all females (1/5 very slight and 4/5 slight). There was a multifocal very slight hyperplasia of pneumocytes type 2 (alveolar bronchiolo-alveolar hyperplasia) in all males and females. In the lung interstitium (perivascular and at the terminal bronchi), a mononuclear cell infiltration was observed in all males (3/5 very slight and 4/5 slight) and all females (all very slight). In the bronchus-associated lymphoid tissues (BALT), a multifocal very slight accumulation of particle-laden macrophages was observed in 4/5 males and 2/5 females and a very slight diffuse increase of lymphocytes was seen in 1/5 males and 2/5 females.
In the lung associated lymph nodes (LALN), there as a multifocal slight accumulation of particle-laden macrophages in all males and females as well as diffuse increase of lymphocytes in all males (4/5 slight and 1/5 moderate) and all females (3/5 slight and 2/5 moderate).
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE FLUID ANALYSIS:
- 1.5, 6 and 24 mg/m³: %macrophages were statistically significant and dose-related decreased in both sexes (68.10 - 32.44 %; p<0.001) compared to controls (approx. 99.60 %). Also, %PMN were statistically significant and dose-related increased in both sexes (31.40 - 63.50 %; p<0.001) compared to controls (0.00 - 0.50 %).
- 6 and 24 mg/m³: leukocyte concentrations (10^6 cells/ml) were statistically significant increased in both sexes (1.00 - 1.25; 0.001 - 0.05) compared to controls (approx. 0.16*10^6 cells/ml). In addtion, lactate dehydrogenase (LDH: 338.8 - 479 U/L), ß-Glucuronidase (BGL: 4.02 - 7.66 U/L) and total protein (TP: 358.2 - 517.4 mg/L) levels were statistically significant and dose-related increased in both sexes compared with the control group (LDH: 84.4 - 98.4 U/L; BGL: 0.62 - 0.80; TP: 112.8 - 123.6 mg/L; p<0.01). For more detailed information please refer to the field "Overall remarks, attachments".
Details on results:
CLINICAL SIGNS:
All animals tolerated well the exposure to the test item at all concentrations. No clinical observations outside the normal range were recorded.

MORTALITY:
All animals survived the study.

BODY WEIGHT AND WEIGHT CHANGES:
Statistically significant changes were not observed in the treatment groups as compared to controls.

HISTOPATHOLOGICAL FINDINGS:
control: in the laryngopharynx, the lung-associated lymph nodes, the nasal cavity, the nasopharynx and the trachea, no pathologic findings were observed. In the lung, in 3/5 males and 2/5 females no visible lesions were found. In 2/5 males and 3/5 females, a multifocal very slight perivascular granulocytic cell infiltration was observed. In the larynx, 2/5 male and 2/5 female rats exhibited a very slight subepithelial mononuclear cell infiltration. All lesions represent commonly found background lesions in the respiratory tract of this rat strain.
Dose descriptor:
LOAEC
Effect level:
1.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other:
Critical effects observed:
not specified
Conclusions:
Wistar rats were exposed to 0.125, 6 and 24 mg/m³ High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix for 6 hours per day, 5 days/week for 14 days by nose-only inhalation. A clean air control group run concurrently.

This study was conducted as a dose range finding study to investigate the toxicity of Pigment 4 using clinical observation, gross pathology, histopathology and bronchoalveolar lavage fluid (BALF) analysis.

All test animals survived during the study, and effects indicating systemic toxicity were not observed. The Body weight development did not show any statistically significant changes as compared to concurrent controls. Lung weights showed statistically significant increases in the mid- and high-dose groups in comparison to the clean air treated control group.
The histopathological examination showed exposure-related adverse findings within the lung: alveolar infiltration of granulocytic cells, alveolar granular material and interstitial mononuclear cell infiltration. The incidence of these findings was increased statistically in the high-dose group.
The accumulation of particle-laden macrophages within the alveoli, the bronchus-associated lymphoid tissue and the lung-associated lymph nodes, the increased amount of alveolar macrophages, the hyperplasia of pneumocytes type 2 and the increased numbers of lymphocytes in the bronchus-associated lymphoid tissue and the lung-associated lymph nodes showed adaptive findings.
Furthermore, the bronchoalveolar lavage fluid (BALF) analysis showed highly increased PMN levels with high statistical significance as compared to controls in all dose groups (ranging approx. from 31% - 64% (males) and 34 - 67 % (females)). In addition, the biochemical parameters mirrored the PMN data for the mid- and high-dose treated groups with lower significance in comparison to controls for lactate dehydrogenase, ß-glucuronidase and total protein. The P4 low-dose group did not show statistical relevant changes.

The Lowest Observed Adverse Effect Concentration (LOAEC) in this study was estimated to be 1.5 mg/m³, based on the PMN values analysed in BALF.
Based on this dose-range finding study a dose scheme of 0.25, 1 and 4 mg pigment 4 per m³ is proposed for the subsequent 90-day nose-only inhalation study (concentration space factor: 4).
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-08-04 to 2021-02-17
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 other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
2018-06-25
Deviations:
yes
Remarks:
Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional in guideline)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2018-11-22
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on species / strain selection:
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55% ± 15%
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.15 - <= 1.82 µm
Remarks on MMAD:
MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: parameters were recorded by 20-minute means. The were set at 22°C ± 2°C for temperature and 55% ± 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to or exactly the target concentrations.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- see above ("Details on inhalation exposure")
The target aerosol concentrations of 0.125, 0.5 and 2 mg/m³ High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix were achieved exactly, i.e. to 100% in each group.
Duration of treatment / exposure:
13 weeks (65 exposure days)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.125 mg/m³ air (analytical)
Remarks:
SD: ± 0.01 mg/m³; 0.02 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
0.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.04 mg/m³; 0.07 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD: ± 0.08 mg/m³; 0.3 mg/lung (calculated total dose using MPPD v3.04)
No. of animals per sex per dose:
10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502; please refer to the study record "s_Creutzenberg_2022" in the IUCLID section 7.2.4) and the 14-day dose-range finding (DRF B) study (Fraunhofer ITEM no. 02 N 20 514; please refer to the study record "s_Creutzenberg_2022_14day" in section 7.5.2).
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.125, 0.5 and 2 mg/m³ were selected to achieve lung burden at the highest concentration that is at or above the lung overload conditions, i.e. impaired lung clearance. The test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.3% (rel. density=2.5, MMAD/GSD=1.8 µm/1.5).
This deposited fraction was used to calculate the total deposited mass, using the following input parameters:
Morphometry: Semi-symmetric Long Evans
Example for deposited mass at 0.125 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.125 mg/m³ x 4.3% = 0.03 mg/lung
Example for deposited mass at 0.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.5 mg/m³ x 4.3% = 0.10 mg/lung
Example for deposited mass at 2 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2 mg/m³ x 4.3% = 0.4 mg/lung
Retained massat 2 mg/m³: approx. 0.3 mg/lung
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day (with the exception of weekends and public holidays: once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: 1 day before treatment and twice a week in the first 4 and once a week thereafter throughout the study for all animals

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was determined for each group on a weekly basis.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water consumption was determined for each group on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: red blood cells (RBC), haemoglobin (HB), haematocrit (HCT), reticulocytes (RET), mean cell volume (MCV), mean haemoglobin/erythrocyte (MCH), mean haemoglobin concentration/erythrocyte (MCHC), prothrombin time (PT), thromboplastin time (TP), total white blood cells (WBC), differential white cell count (% and absolute), platelets (PTL)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), urea, triglycerides, total bilirubin, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB, glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K), phosphorous (P), chloride (CL)

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 90 days post-exposure
- Dose groups that were examined: all
- Number of animals: 5 (90 days post-exposure) - 10 (1 day post-exposure) per sex and dose group
- Parameters examined: total cell count, differential cell count (macrophages, neutrophils, eosinophils, lymphocytes; a total of 400 leukocytes per rat were evaluated), LDH, β-glucuronidase, and total protein

LUNG BURDEN: Yes (determination of lung:body weight ratio before and after treatment as well as chemical analysis)
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
- Chemical analysis: after sacrifice the lungs were prepared by freeze drying (-20 °C for approx. 48 h), plasma ashing (approx. 48 h, cool plasma conditions), and (wet) micowave digestion (H2SO4 (96 % and HNO3, HF)).
Please refer to IUCLID section 7.1.1 "k_Creutzenberg_2022_lung burden" for more details on the method of lung burden analysis.
Sacrifice and pathology:
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 4 (High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix) high dose group after 90 days nose-only inhalation.
These organs were as follows: adrenal glands, aorta, bone marrow (femur and sternum), brain (cerebrum, cerebellum, pons/medulla), coagulating glands, epididymis, esophagus, femur with knee joint, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx, lung (left main lobe), liver, lymph nodes (lung-associated, mandibular, mesenteric), mammary gland, nasal cavity, olfactory bulb (in situ), ovaries, oviducts, pancreas, parathyroid glands, peripheral (sciatic) nerve, pituitary gland, prostate, salivary glands (mandibular, parotid, sublingual), seminal vesicles, skeletal muscle (biceps femoris), skin, spinal cord (cervical, thoracic and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterine cervix, uterus, vagina.
The following respiratory tract organs of animals 101-110 and 201-210 of group 1 and 7, respectively, were examined histopathologically: Nasal and paranasal cavities, pharynx (Laryngopharynx/nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology was performed in 10 animals per sex of the control and high dose group at day 1 post-exposure. Additionally, histopathological examination of animals of both sexes of the low and intermediate dose group was performed.
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups were compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings were done with the two-tailed Fisher test by the PROVANTIS system.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females, statistically significant dose-dependent increases were observed for leukocytes (WBC), segmented (SEGC) and banded (BANC) neutrophils in the mid and high dose groups (calculated and percentual values) (please refer to: 'Attached background material'):
- The WBC was statistically significantly increased in males and females of the mid-dose (+35.5% and +42.2%, respectively) and high-dose (+35.1% and +35.9%, respectively).
- The BANC was statistically significantly increased in males and females of the mid-dose (+129.9% and +137.6%, respectively) and high-dose (+121.4% and +152.7%, respectively).
- The SEGC was statistically significantly increased in males and females of the mid-dose (+92.3% and +104.7%, respectively) and high-dose (+121.1% and +125.6%, respectively).
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
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):
Absolute and relative lung wet weights resulted dose-dependently in statistically significant increases in the mid- and high-dose groups of both sexes (please refer to: ‘Attached background material’):
- One day after the last exposure, males exposed to 2 mg/m³ showed an increase of 92.5% in absolute lung wet weight. The relative lung weight was increased by 21% and 93.5% in the mid- and high-dose groups, when compared to the control group. At post-exposure day 92, the absolute lung weight was increased by 113.2%. The relative lung weight was increased by 125.3%.
- One day after the last exposure, females exposed to 0.5 and 2 mg/m³ showed statistically significant increases in the absolute lung wet weight (+24.0% and +86.6%). The relative lung weight was increased by 19.4% and 81% in the mid- and high-dose groups, when compared to the control group. At post-exposure day 92, the absolute lung weight was increased by 22.9% and 94.8% in the mid- and high-dose groups. The relative lung weight was increased by 20.3% and 89.3%.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon necropsy, enlarged lung-associated lymph nodes (LALN) were observed dose-dependently in the mid and high dose groups of both sexes (please refer to: ‘Attached background material’). This is a particle-specific lung clearance pathway. Lungs showed typical dose-dependent discolourations caused by the test item.
- One day after the last exposure, the LALN were found to be enlarged in 1/20, 8/20, 20/20, and 20/20 males exposed to 0, 0.125, 0.5, and 2 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the LALN were found to be enlarged in 2/10, 5/10, 9/10, and 10/10 animals, respectively. At post-exposure day 90, the LALN were enlarged in 0/5, 3/5, 5/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 4/5, 5/5, and 5/5 in the control, low, intermediate, and high dose group, respectively.
- One day after the last exposure, the lungs showed discoloured areas in 0/20, 3/20, 5/20, and 20/20 males exposed to 0, 0.125, 0.5, and 2 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the lungs showed discoloured areas in 1/10, 1/10, 2/10, and 9/10 animals, respectively. At post-exposure day 90, the lungs showed discolouration in 0/5, 3/5, 0/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 0/5, 0/5, and 5/5 in the control, low, intermediate, and high dose group, respectively.


Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings related to the inhalation of the test item were detected within the lung, the nasal cavity and the lung-associated lymph nodes.

- Animals exposed to 0.125 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 5/10 male (4 very slight; 1 slight) and 8/10 female rats (8 very slight). Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (10 very slight) and in 10/10 females (10 very slight) as well as in the bronchus-associated lymphoid tissue in 1/10 males (1 very slight) and in 1/10 females (1 very slight). In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 4/10 males (2 very slight; 1 slight; 1 moderate) and in 1/10 females (1 very slight). There was a multifocal very slight bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 1/10 males and a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 1/10 males.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 1/10 males (1 moderate) and in 1/10 females (1 very slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi.

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 2/10 males (2 very slight) and in 9/10 females (8 very slight; 1 slight). In addition, 1 male (1 slight) and 3 females (3 slight) showed a diffuse increased cellularity of lymphocytes.
•These lesions correlated with a macroscopically observed enlargement in all animals.

- Animals exposed to 0.5 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 10/10 male (4 very slight; 6 slight) and 10/10 female rats (6 very slight; 4 slight). In addition, in the lung alveoli, multifocal extracellular material was seen in 5/10 male (4 very slight; 1 slight) and 5/10 female rats (5 very slight). This extracellular material appeared eosinophilic partially granular and was interpreted to be consisting of cell debris and surfactant admixed with particles. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (1 very slight; 9 slight) and in 10/10 females (3 very slight; 7 slight) as well as in the bronchus-associated lymphoid tissue in 8/10 males (5 very slight; 3 slight) and in 7/10 females (2 very slight; 5 slight). This lesion correlated with a macroscopically observed pale white multifocal discoloration of up to 1mm in diameter in the lung. In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 10/10 males (1 very slight; 8 slight; 1 moderate) and in 9/10 females (4 very slight; 4 slight; 1 moderate). There was a multifocal very slight bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 2/10 males and in 3/10 females as well as a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 1/10 males.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 7/10 males (6 very slight; 1 slight) and in 5/10 females (5 very slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi. In addition to the accumulation of particle-laden macrophages in the BALT, diffusely the lymphocytes were increased in numbers very slight in 1/10 males and 2/10 females.

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (1 very slight; 1 slight; 8 moderate) and in 10/10 females (3 slight; 5 moderate; 2 severe). This was accompanied by a central necrosis of the accumulation of particle-laden macrophages in 3/10 males (2 very slight; 1 slight) and 3/10 females (2 slight; 1 moderate). In addition, 8/10 males (4 slight; 4 moderate) and 9 females (3 very slight; 4 slight; 2 moderate) showed a diffuse increased cellularity of lymphocytes.
• These lesions correlated with a macroscopically observed enlargement in all animals.

- Animals exposed to 2 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 10/10 male (7 slight; 3 moderate) and 10/10 female rats (1 very slight; 6 slight; 3 moderate). In addition, in the lung alveoli, multifocal extracellular material was seen in 10/10 male (1 slight; 9 moderate) and 10/10 female rats (1 slight; 8 moderate; 1 severe). This extracellular material appeared eosinophilic partially granular and was interpreted to be consisting of cell debris and surfactant admixed with particles. The more severe cases represent a beginning lipoproteinosis. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (4 slight; 6 moderate) and in 10/10 females (7 slight; 3 moderate) as well as in the bronchus-associated lymphoid tissue in 10/10 males (1 very slight; 6 slight; 3 moderate) and in 10/10 females (5 very slight; 4 slight; 1 moderate). This lesion correlated with a macroscopically observed pale white multifocal discoloration of up to 1mm in diameter in the lung. In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 10/10 males (3 very slight; 5 slight; 2 moderate) and in 10/10 females (2 very slight; 8 slight). There was a multifocal bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 10/10 males (6 very slight; 4 slight) and in 10/10 females (5 very slight; 5 slight) and a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 5/10 males and in 3/10 females.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 10/10 males (1 very slight; 9 slight) and in 10/10 females (4 very slight; 6 slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi. In addition to the accumulation of particle-laden macrophages in the BALT, diffusely the lymphocytes were increased in numbers in 8/10 males (6 very slight; 2 slight) and 6/10 females (5 very slight; 1 slight).

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (3 moderate; 7 severe) and in 10/10 females (3 moderate; 7 severe). This was accompanied by a central necrosis of the accumulation of particle-laden macrophages in 10/10 males (6 very slight; 4 slight) and 9/10 females (3 very slight; 4 slight; 2 moderate). In addition, 9 males (1 very slight; 4 slight; 3 moderate; 1 severe) and 9 females (2 very slight; 3 slight; 4 moderate) showed a diffuse increased cellularity of lymphocytes.

• In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 1/10 females in the nose-associated lymphoid tissue (NALT).

Please refer to: ‘Attached background material’ for statistical intergroup comparison and detailed listing.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE FLUID (BALF):
- At days 1 and 90 post-exposure statistically significant and dose-dependent increases of polymorphonuclear neutrophils (PMN) were detected in all treatment groups (please refer to: ‘Attached background material’). The PMN percentages (in the range from 25% to 55%, males - 27% to 50%, females in all dose groups) demonstrate a strong inflammatory potential as compared to clean air control data (3-4% PMN) at the given low aerosol concentrations.
- At day 1 post-exposure, for lactic dehydrogenase, ß-glucuronidase and total protein statistically significant and dose-dependent increases were detected in the mid (but ß-glucuronidase) and high-dose groups both sexes; after 90 days of recovery, however, still statistically significant increases in the high dose group:
- Lactic dehydrogenase activity: At day 1 post-exposure, lactic dehydrogenase activity was 3.7- and 7.6-fold increased in the male mid- and high-dose group, respectively. In females, the lactic dehydrogenase activity was 3.4- and 8.9-fold increased, respectively. At post-exposure day 92, females of the high dose group showed a 5.3-fold increased lactic dehydrogenase activity as compared to the control group.
- ß-glucuronidase activity: At day 1 post-exposure, the ß-glucuronidase activity was 31.5-fold increased in males of the high-dose group, when compared to the negative control group value. In females, the ß-glucuronidase activity was 32.6-fold increased over controls. At post-exposure day 92, females of the high dose group showed a 15.1-fold increased ß-glucuronidase activity as compared to the control group.
- Total protein concentration: at day 1 post-exposure, the total protein concentration was 2.4- and 4.8-fold increased in the male mid- and high-dose group, respectively. In females, the total protein concentration was 2.2- and 5.7-fold increased, respectively. At post-exposure day 92, females of the high dose group showed a 4.4-fold increased total protein concentration as compared to the control group.
Details on results:
FOOD CONSUMPTION:
- Some observed statistically altered values were considered as incidental findings as these effects were without a clear dose-dependency.

WATER CONSUMPTION:
- Some observed statistically altered values were considered as incidental findings as these effects were without a clear dose-dependency.

HAEMATOLOGICAL FINDINGS:
- One day after the end of exposure, females exposed to 0.125 mg/m³ showed a marginal but statistically significantly increased haematocrit value (+4.3%). However, no such effect was observed at higher doses. Thus, the finding is considered to be of an incidental nature and without toxicological relevance. Moreover, the thromboplastin time showed a mild but statistically significant reduction in males exposed to 0.5 (-8.9%) and 2 mg/m³ (-7%). The decrease was, however, not dose dependent and is considered to be not toxicologically relevant.

CLINICAL BIOCHEMISTRY FINDINGS:
- Animals exposed to 0.125 mg/m³: In females, the total bilirubin concentration was statistically significantly decreased (-19.3%) on post-exposure day 1 (please refer to: ‘Attached background material’). Moreover, total protein and albumin levels were statistically significantly increased (+5.4% and 4.1%, respectively) on post-exposure day 1. However, no such effects were observed in either higher dose groups or in males. Thus, these effects are considered to be incidental and without toxicological relevance.

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- Incidental increases were observed in the absolute weight of the left ovary of females exposed to 0.5 mg/m³ (post-exposure day 1; +26.2%), absolute and relative weight of the thymus of males exposed to 0.125 mg/m³ (post-exposure day 92; +39.7% and +32.6%, respectively), absolute and relative weight of the left adrenal of females exposed to 0. 5 mg/m³ (post-exposure day 92; +36.8% and +33.9%, respectively), absolute weight of the uterus of females exposed to 0.125 mg/m³ (post-exposure day 92; +14.3%), as well as increased relative weight of the left and right kidneys of females exposed to 0.5 mg/m³ (post-exposure day 92; +13% and +11%, respectively). These alterations were only slight in magnitude, not consistent within/between animals, not dose-dependent, and without histopathological correlates. Thus, these effects are considered to be incidental and without toxicological relevance.

GROSS PATHOLOGICAL FINDINGS:
- Other test item- or dose-related macroscopical findings were not detected. Only some incidental macroscopic observations were obtained (please refer to: ‘Attached background material’).

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- Clean air control: In one animal of the control group, a lymphoma of the hematolymphoid system with infiltration of lymphoma cells into the bone marrow, liver, lung-associated, mandibular and mesenteric lymph nodes, and spleen was observed. This correlated with macroscopically observed enlarged spleen and lung-associated lymph nodes. In addition, single lesions were seen in different other organs, which represented commonly found background lesions in this rat strain.
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.
- Animals exposed to 0.125 and 0.5 mg/m³: In the nasal cavity and trachea no lesions were seen.
- Animals exposed to 2 mg/m³: the occurrence of very slight multifocal hyaline droplets in 2 males was considered to be unrelated to the exposure.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
- Lung weights: In both sexes, no statistically significant changes as compared to concurrent controls.
Key result
Dose descriptor:
LOAEC
Effect level:
0.125 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: BALF
Critical effects observed:
not specified

Please refer to IUCLID section 7.1.1 "k_Creutzenberg_2022_lung burden" for more details on results of the lung burden analysis.

Conclusions:
In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.125, 0.5 and 2 mg/m³ of high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix.

All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption.
Haematology showed statistically significant dose-dependent increases for leukocytes, segmented and banded neutrophils in the mid and high dose groups, being concomitant with lung inflammation. The lung wet weight was dose-dependently in statistically significantly increased in the mid and high dose groups. The BALF analyses revealed persistent, statistically significant and dose-dependent increases of polymorphonuclear neutrophils (PMN) in all treatment groups demonstrating, based on the effect magnitude, a strong inflammatory potential. Moreover, lactate dehydrogenase (LDH), ß-glucuronidase and total protein showed persistent (in females), statistically significant and dose-dependent increases in the mid (but ß-glucuronidase) and high dose groups.
Adverse findings, as revealed by histopathology, representing very slight to moderate alveolar granulocytic cell infiltration, very slight to severe alveolar extracellular material, very slight to moderate lung interstitial mixed inflammatory cell infiltration, and very slight to moderate necrosis of particle-laden macrophages in the lung-associated lymph nodes, were detected within the investigated high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix mid- and high-dose group and partially in the low dose group.

Under the conditions of this test, a LOAEC of 0.125 mg/m³ was derived for both sexes on the basis of the inflammatory response seen in the lung (BALF and histopathology).
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
acute toxicity: other routes
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2020-02-24 to 2020-03-02
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This study was conducted as a dose range finding study, with limited study design focussing on assessing local effects of five different inorganic pigment substances. The non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess acute inhalation toxicity. The investigated mechanistic parameters (bronchoalveolar lavage (BAL) fluid analyses have no direct value for fullfilling data requirements under the REACH regulation.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: OECD guideline No. 412 (28-day (subacute) inhalation toxicity study)
Version / remarks:
2018-06-15
Deviations:
yes
Remarks:
The study was conducted as range-finding study. Therefore, it has a limited study design.
Principles of method if other than guideline:
Creutzenberger, O. (2020), exposed female Wistar rats to the the test item to doses of 0.2, 0.8 and 3.2 mg by intratracheal instillation (total dose was instilled on two consecutive days) to investigate the lung toxicity potential with a bronchoalveolar lavage (BAL) analysis.
GLP compliance:
no
Remarks:
The investigations in DRF A were conducted under non-GLP conditions because of the screening, dose-range finding nature of the experiments. However, the experimental procedures followed the GLP rules (e.g. use of SOPs and documentation/archiving).
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 8 weeks old
- Weight at study initiation: approx. 186 g
- Housing: housed in Makrolon® (polycarbonate) cages type Ill, four rats per cage in the treated groups and three rats per cage in the vehicle control group; absorbing softwood ('ssn BK 8-15') bedding material.
- Diet: commercial chow in pellet form (ssniff”V1534; supplier: ssniff Spezialdiäten GmbH, Soest, Germany)
- Water: tap water
- Acclimation: approximately one week the animals were allowed to adjust and become acclimatized to the Fraunhofer ITEM environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
other: intratracheal instillation
Vehicle:
other: saline solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in vehicle, each aliquot in a volume of 0.3 mL. After gentle shaking all samples were sonicated for 2 minutes to guarantee a homogeneous suspension. After sonication samples were shaken again to perpetuate the homogeneity until administration to the animals.
The total dose was instilled in two aliquots on two consecutive days to achieve a homogeneous distribution of the test material in lungs.
Doses:
0.08, 0.25 and 1 mg
No. of animals per sex per dose:
4 females in intreated group; 6 females in control group
Control animals:
yes
Remarks:
vehicle control
Details on study design:
- Duration of observation period following administration: 3 days
- Frequency of observations and weighing: all animals were clinically observed in their cages at least twice a day. Before sacrifice, they were inspected outside their home cages and carefully examined for clinical symptoms, i.e. abnormalities concerning their general condition. On the treatment days, the animals were clinically observed before and after treatment.
Individual body weight was recorded on day -1 before treatment and on day 3 at sacrifice for all animals.
- Necropsy of survivors performed: yes, all animals were subjected to a complete necropsy.

ORGAN WEIGHT:
the lungs were weighed, and the relative lung weight was calculated.

BRONCHOALVEOLAR FLUID (BALF) ANALYSIS:
analysis was performed in all rats 3 days after the last instillation. The total cell count and differential cell count were determined. The method of Henderson et al. (1987) was used with minor modifications.*
Following preparation, the lungs were lavaged with saline using two lavages of 4 ml (if half lung will be used only: 2 ml). The pooled lavage fluid was collected in calibrated tubes and the harvested volume was recorded. Until processing the lavage fluid was kept on ice. Leukocyte concentration of the lavagate was determined using a counting chamber and two cytoslides were prepared with a cytocentrifuge for differential cell count (macrophages, neutrophils, eosinophils, lymphocytes).

*References:
Henderson, R.F., Mauderly, J.L. Pickrell, J.A., Hahn, R.F., Muhle, H., and Rebar, A.H. (1987): Comparative study of bronchoalveolar lavage fluid: Effect of species, age and method of lavage. Exp. Lung Res. 13, 329 342.
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Body weight and lung wet weight data were calculated using a two-sample t-test assuming equal variances. BAL data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test.
Remarks on result:
not determinable because of methodological limitations
Mortality:
All animals survived the study.
Clinical signs:
All animals tolerated well the exposure to the test item at all concentrations. No clinical observations outside the normal range were recorded.
Body weight:
Body weight development did not show any statistically significant changes as compared to concurrent controls.
Gross pathology:
Upon necropsy, test item- or dose-related macroscopical findings were observed, like enlarged lung associated lymph nodes (LALN) or white areas (Ø < 1 mm in diameter) in some pigment powder treated groups. Some white areas were shown in the lung of animals in the high-dose group, as well as in the lung of one animal each of the low- and mid-dose groups. The LALN were enlarged slightly to moderately in the mid-dose group and moderately to highly in the high-dose group.
Other findings:
ORGAN WEIGHT:
The absolute lung weight was statistically significant increased in the mid- and high-dose group (1.127 ± 0.081 g; 1.189 ± 0.034 g; p< 0.001 - 0.01) compared to the controls (0.985 ± 0.051 g), but the relative lung weight was not increased.

BRONCHOALVEOLAR LAVAGE FLUID ANALYSIS:
At day 3 post-treatment, statistically significant and treatment-related decrease was observed in the percentages of macrophages (82.13 ± 3.09 %; 60.38 ±1.16 %) and an increase in %PMN (16.69 ± 2.44 ; 38.00 ± 1.93 %) in the mid- and high-dose group compared to the controls (98.88 ± 1.41 % and 1.04 ± 1.25 %; p<0.001). The low-dose animals showed %macrophage and %PMN values in the range of the control animals (98.75 ± 0.79 %; 1.06 ± 0.63 %). Also in the high-dose group, the percentage of lymphocytes (1.63 ± 0.88 %; p<0.01) were statistically significant and dose-related increased compared to the controls. The low- and mid-dose animals showed %lymphocytes in the range of the control animals (0.08 ± 0.20 %).
Conclusions:
Creutzenberg, O. (2020), exposed female Wistar rats to high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix to doses of 0.08, 0.25 and 1 mg by intratracheal instillation (total dose was instilled on two consecutive days) to investigate the lung toxicity potential with a bronchoalveolar lavage (BAL) analysis. A vehicle control group was run concurrently.

This study was conducted as a dose range finding study, with limited study design focussing on assessing local effects of five different inorganic pigment substances.
The highest dose in this instillation experiment was assumed to lead to an overload condition (by way of read-across from other PSLT substances), which leads to an inflammatory response inter alia characterised by an increase of granulocytic cells.

Upon necropsy, test item- or dose-related macroscopical findings were observed, like enlarged lung associated lymph nodes (LALN) or white areas (Ø < 1 mm in diameter) in some pigment powder treated groups.

The bronchoalveolar lavage fluid analysis showed statistically significant and treatment-related decrease in the percentages of macrophages (82%; 60%) and an increase in %PMN (16.7%; 38%) in the mid- and high-dose group compared to the controls (98.9% and 1.04%) at day 3 post-treatment. The low-dose animals showed %macrophage and %PMN values in the range of the control animals (98.8%; 1.06%). Also in the high-dose group, the percentage of lymphocytes (1.63 %; p<0.01) were statistically significant and dose-related increased compared to the controls. The low- and mid-dose animals showed %lymphocytes in the range of the control animals (0.08 %).

On the basis of the results obtained in the BALF analysis after in vivo instillation of five different inorganic pigments combined with deposition modelling (using the MPPD model), the pigment with the highest reactivity in the respiratory tract was used for a subsequent 14-day inhalation dose-range finding study.
For the other four inorganic pigments, the concentrations for the main 90-day study was based on the results obtained in this instillation experiments combined with deposition modelling (using the MPPD model).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Objective of study:
absorption
Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 413 (90-day (Subchronic) Inhalation Toxicity Study)
Version / remarks:
2018-06-25
Deviations:
yes
Remarks:
Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional in guideline)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2018-11-22.

Test material

Constituent 1
Chemical structure
Reference substance name:
oxo[(oxoferrio)oxy]iron; silanedione
EC Number:
701-304-2
Cas Number:
1353091-50-5
Molecular formula:
(SiO2)(Fe2O3)
IUPAC Name:
oxo[(oxoferrio)oxy]iron; silanedione
Test material form:
solid: particulate/powder
Details on test material:
- Name: Colorante Rojo
- OLD EC Name: Reaction mass of fumes, silica and diiron trioxide
- NEW EC Name: High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix
- Substance type: inorganic pigment
- Physical state: solid, red powder, odourless
- Storage condition of test material: at room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light.
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: Crl:WI (Han)
Details on species / strain selection:
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet (ad libitum): commercial chow in pellet form (ssniff “V1534”; supplier: ssniff Spezialdiäten GmbH, Soest, Germany).
- Water (ad libitum): tap water
- Acclimation period: approx. one week the animals were allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats were trained to the 6-hour restraint in nose-only tubes.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55% ± 15%
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
inhalation: aerosol
Vehicle:
unchanged (no vehicle)
Remarks:
filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: the particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: parameters were recorded by 20-minute means. The were set at 22°C ± 2°C for temperature and 55% ± 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: the MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to or exactly the target concentrations.
- Samples taken from breathing zone: yes
Duration and frequency of treatment / exposure:
13 weeks (65 exposure days); 6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0.125 mg/m³ air (analytical)
Remarks:
SD: ± 0.01 mg/m³; 0.02 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
0.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.04 mg/m³; 0.07 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD: ± 0.08 mg/m³; 0.3 mg/lung (calculated total dose using MPPD v3.04)
No. of animals per sex per dose / concentration:
15 males: 5 males (1 day recovery), 5 males (28 days recovery), and 5 males (90 days recovery)
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
none
Details on study design:
- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.125, 0.5 and 2 mg/m³ were selected to achieve lung burden at the highest concentration that is at or above the lung overload conditions, i.e. impaired lung clearance. The test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.3% (rel. density=2.5, MMAD/GSD=1.8 µm/1.5).
This deposited fraction was used to calculate the total deposited mass, using the following input parameters:
Morphometry: Semi-symmetric Long Evans
Example for deposited mass at 0.125 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.125 mg/m³ x 4.3% = 0.03 mg/lung
Example for deposited mass at 0.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.5 mg/m³ x 4.3% = 0.10 mg/lung
Example for deposited mass at 2 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2 mg/m³ x 4.3% = 0.4 mg/lung
Retained massat 2 mg/m³: approx. 0.3 mg/lung
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: lungs
- Time and frequency of sampling: 1, 28, and 90 days after the 90-day exposure period

ANALYTICAL METHOD
- Complete description including: lungs of 5 male rats in all exposure groups were subjected to a chemical analysis to verify the predicted retained mass of the test items after 90 days of inhalation +1; +28 and + 90 recovery days. For recovery days +1 and +28 whole lungs were available. On +90 days the right lung lobe was available for analysis. Here a conversion factor of 1.67 was applied to extrapolate the lung burden to the whole lung. Between animal sacrifice and sample preparation samples were stored at -20 °C. Prior to microwave digestion lung tissue samples underwent a freeze-drying step (approx. 48 h), followed by plasma ashing (approx. 48 h, cool plasma conditions). Samples processed this way underwent microwave digestion. H2SO4 (1 mL) and HNO3 (4 mL) were added to sample in a PP tube and left sitting at room temperature for 2 h. Afterwards water (2.5 mL) was added and samples left to cool before adding HF (1 mL). After 16 h the sample volume was made up to 50 mL with deionised water. After appropriate dilution (see raw data) with deionised water samples were analysed by ICP-MS. Since hydrofluoric acid (HF) was used for digestion, H3BO3 was added during sample dilution (see raw data). Quantification was achieved against matrix matched standards. To ensure validity of the analysis data, samples were bracketed by QC standards.

System: icap Q (ThermoScientific) or icap TQ in single quadrupole mode (ThermoScientific);
Autosampler: Cetac ASX 520 or ESI 4DX;
Interface: High matrix;
Mode: KED (Helium);
Plasma [W]: 1.550;
Spray chamber: Cyclonic;
Number of main runs: 5;
Analytes (m/z) (Qualifier; Quantifier): 54Fe; 56Fe; 57Fe;
Internal standards (m/z): Chromium: 45Sc, 74Ge
Statistics:
Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.

Results and discussion

Preliminary studies:
A dose range finding study as acute toxicity study by intratracheal instillation was conducted. For further information please refer to the study record "s_Creutzenberg_2022" in IUCLID section 7.2.4. In addition, a 14-day repeated dose inhalation toxicity study was conducted which can be found in the study record "Creutzenberg_2022_14day" in IUCLID section 7.5.2.
Main ADME results
Type:
absorption
Results:
lung burden with test item of lungs after 1, 28, and 90 days after the 90-day exposure period:
- control: 1880, 1831 & 1449 μg/lung
- 0.125 mg/m3: 2053, 2066 & 1721 μg/lung
- 0.5 mg/m3: 2262, 2683 & 1954* μg/lung
- 2 mg/m3: 3527, 4496 & 4019 μg/lung
*n=4

Toxicokinetic / pharmacokinetic studies

Details on absorption:
For detailed information of absorption in lung tissue please refer to the filed "overall remarks, attachments".
Details on distribution in tissues:
not examined
Details on excretion:
not examined

Metabolite characterisation studies

Metabolites identified:
not measured

Enzymatic activity

Enzymatic activity measured:
not examined

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
not examined

Any other information on results incl. tables

It was not possible to calculate clearance half-times.

Applicant's summary and conclusion

Conclusions:
Male rats were exposed to concentrations of 0.4, 1.5 and 6 mg High-temperature calcination products of diiron trioxide and amorphous silica (Pigment 4) resulting in a glassy silica matrix/m³ air for 6 hours per day, 5 days/week for 90 days via nose-only inhalation. The lung burden with high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix were determined 1, 28 and 90 days after the 90-day exposure period.

One day, 1 month and 3 months after end of exposure, in the low dose groups 0.17, 0.24 and 0.27 mg/lung, in the mid dose groups 0.38, 0.85 and 0.51 mg/lung, and in the high dose groups 1.65, 2.67 and 2.57 mg/lung of the test item (Pigment 4) were determined, respectively. The clearance half-times of the test-item were not calculated. The retained masses determined at day 1 post-exposure are in all dose groups higher than the predicted values calculated under the assumption of physiological clearance conditions. During the recovery period a lung clearance effect was not observed in any dose group. The conclusion is that the high inflammatory potential of the test item reduced the clearance capacity and thus induced an overproportional increase of retained test item in all dose groups. This effect may be enhanced by an altered breathing frequency resulting in higher minute volumes. The lung burden increase from day 1 to day 28 can be explained assuming biological variation (group size: N=5). Consequently, to the reported data, it was not possible to calculate clearance half-times.