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EC number: 701-304-2 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Acute Toxicity: other routes
Administrative data
- 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.
Cross-reference
- Reason / purpose for cross-reference:
- reference to other 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
- 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).
Please refer to IUCLID section 7.1.1 "k_Creutzenberg_2022_lung burden" for more details on results of the lung burden analysis.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
Materials and methods
Test guideline
- 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
Test material
- 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
Constituent 1
- 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
Test animals
- 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
Administration / exposure
- 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.
Results and discussion
Effect levels
- 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 %).
Applicant's summary and conclusion
- 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).
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