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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-25 to 2021-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-25 to 2021-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Objective of study:
absorption
other: lung clearance
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.
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
Species:
rat
Strain:
Wistar
Remarks:
Wistar (Han)
Sex:
male
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. 250 g for males and approx. 175 g for females
- Housing: housed in polycarbonate Makrolon cages type III with 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 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
Vehicle:
unchanged (no vehicle)
Remarks:
filtered air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

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 the target concentrations. Permanent control of the aerosol concentrations is guaranteed by photometers
- Samples taken from breathing zone: yes
Duration and frequency of treatment / exposure:
13 weeks (65 exposure days); 6 hours/day, 5 days/week
Dose / conc.:
0.4 mg/m³ air (analytical)
Remarks:
SD: ± 0.02 mg/m³; 0.03 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.05 mg/m³; 0.125 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
6 mg/m³ air (analytical)
Remarks:
SD: ± 0.23 mg/m³; 1.0 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

For the nominal aerosol concentrations of 0.4, 1.5 and 6 mg/m³ the test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.6 % (rel. density=4.5, 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 0.4 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.4 mg/m³ x 4.7% = 0.03 mg/lung
Example for deposited mass at 1.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 1.5 mg/m³ x 4.7% = 0.125 mg/lung
Example for deposited mass at 6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 6 mg/m³ x 4.7% = 1.0 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). H2SO4 (4 mL) and HNO3 (1 mL) were added to sample in a quartz glass vial and subjected to a microwave digest (max. 500 W). Samples were left to cool down and transferred into PP tubes 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 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): 59Co;
Internal standards (m/z): Chromium: 45Sc, 74Ge;
Limit of quantification: < 3.27 µg/lung.
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.
Preliminary studies:
A dose range finding study by intratracheal instillation was conducted. For further information please refer to the study record d_Creutzenberg_2022 in IUCLID section 7.2.4.
Type:
absorption
Results:
Lung burden with cobalt zinc aluminate blue spinel after 1, 28, and 90 days after the 90-day exposure period:
- control: < LOQ;
- 0.4 mg/m3: 0.12, 0.07 & 0.03 mg/lung;
- 1.5 mg/m3: 0.42, 0.27 & 0.17 mg/lung;
- 6 mg/m3: 2.28, 1.67 and 1.39 mg/lung.
Type:
other: lung clearance half-time
Results:
after exposure to 0.4 mg cobalt zinc aluminate blue spinel/m³: 46.5 days
Type:
other: lung clearance half-time
Results:
after exposure to 1.5 mg cobalt zinc aluminate blue spinel/m³: 68.6 days
Type:
other: lung clearance half-time
Results:
after exposure to 6 mg cobalt zinc aluminate blue spinel/m³: 138.6 days
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
Metabolites identified:
not measured
Enzymatic activity measured:
not examined
Bioaccessibility (or Bioavailability) testing results:
not examined
Conclusions:
Creutzenberg, O. (2022), exposed male rats to a concentration of 0.4, 1.5 and 6 mg cobalt zinc aluminate blue spinel/m³ air for 6 hours per day, 5 days/week for 90 days via nose-only inhalation. The lung burden and clearance with cobalt zinc aluminate blue spinel 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 0.12, 0.07 and 0.03 mg/lung, in the mid dose groups 0.42, 0.27 and 0.17 mg/lung, and in the high dose groups 2.28, 1.67 and 1.39 mg/lung of the test item Cobalt Zinc Aluminate Blue Spinel (Pigment 1) were determined, respectively.

The clearance half-times of the test item amounted to 47 and 69 days in the low and mid dose group, respectively, thus were very close to the physiological half-time being approx. 60 days (ECETOC, 2013), or 50.5 days (median over all 5 sub-chronic inhalation toxicity studies, low-dose animals). In the high dose group, a half-time of 139 days was determined, being well above a 2-fold increase as compared to the physiological values of 50.5 or 60 days. The concentration-dependent increase in clearance half-times is indicative for a poorly soluble low toxicity (PSLT) particle, which may lead to a lung overload condition, i.e. impaired clearance in which the deposited dose of inhaled PSLT in the lung overwhelms clearance from the alveolar region leading to a reduction in the ability of the lung to remove particles. The prolongation of the clearance half-time of two or more-fold above the physiological value in the high dose group demonstrates that an overload of particle clearance condition has been reached (Driscoll and Borm, 2020).
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:
Creutzenberg, O. (2020), exposed female Wistar rats to cobalt zinc aluminate blue spinel 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. 182 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:
0.2, 0.8 and 3.2 mg
No. of animals per sex per dose:
4 females in treated 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.
Dose descriptor:
other:
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. In the low- and mid-dose group, the LALN were slightly to moderately enlarged. However, there were not any special findings in the lung of these animals. In the high-dose group, the LALN were slightly enlarged in the animals, and there were some white areas (Ø < 1 mm) in one animal. The lung showed dark blue areas.
Other findings:
ORGAN WEIGHT:
The absolute lung weight was statistically significant increased in the high-dose group (1.138 ± 0.062 g; P< 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 increase in the number of polymorphnuclear neutrophils (PMN: 34.00 ± 4.26 %; P<0.001) were observed in the high-dose treated group compared to the controls (1.04 ± 1.25 %). The low- and mid- dose animals showed %PMN values in the range of the control animals (1.13% ± 0.48 and 2.19% ± 0.97).

Also in the high-dose group, the percentage of macrophages was statistically significant decreased (65.06 ± 4.42 %; P<0.001 %) compared to the controls (98.99 ± 1.41 %).
Conclusions:
Creutzenberg, O. (2020), exposed female Wistar rats to cobalt zinc aluminate blue spinel 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. 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 (up to 2 mm in diameter) in some pigment powder treated groups. The absolute but not the relative lung weights were increased in the animals of the high-dose group compared to controls.

The bronchoalveolar fluid analysis showed a statistically significant (p<0.001) decrease in the percentages of macrophages (65%) and an increase of PMNs (34% ± 4.26) in the high-dose group compared to the controls (99% and 1%). The low- and mid- dose animals showed %PMN values in the range of the control animals (1.13% ± 0.48 and 2.19% ± 0.97).

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

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Cobalt zinc aluminate blue spinel
EC Number:
269-049-5
EC Name:
Cobalt zinc aluminate blue spinel
Cas Number:
68186-87-8
Molecular formula:
Co(x)Zn(1-x)Al2O4 0,1≤x≤0,9
IUPAC Name:
Cobalt zinc aluminium spinel
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Cobalt zinc aluminate blue spinel
- Physical state: Solid, blue powder, odourless
- Structure: spinel
- Storage condition of test material: Kept dry in closed containers
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:
Wistar (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. 250 g for males and approx. 175 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff BK 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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.26 - <= 1.57 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 the target concentrations. Permanent control of the aerosol concentrations is guaranteed by photometers
- 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 0.4, 1.5 and 6 mg/m³ Cobalt Zinc Aluminate Blue Spinel 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
Doses / concentrationsopen allclose all
Dose / conc.:
0.4 mg/m³ air (analytical)
Remarks:
SD: ± 0.02 mg/m³
Dose / conc.:
1.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.05 mg/m³
Dose / conc.:
6 mg/m³ air (analytical)
Remarks:
SD: ± 0.23 mg/m³
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).
- Post-exposure recovery period: 1, 28, and 90 days

For the nominal aerosol concentrations of 0.4, 1.5 and 6 mg/m³ the test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.6% (rel. density=4.5, MMAD/GSD=1.8µm/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 0.4 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.4 mg/m³ x 4.7% = 0.03 mg/lung
Example for deposited mass at 1.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 1.5 mg/m³ x 4.7% = 0.125 mg/lung
Example for deposited mass at 6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 6 mg/m³ x 4.7% = 1.0 mg/lung
Positive control:
none

Examinations

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 (if feeding study): 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 (if drinking water study): 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
- 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 (> 6 hours (0.37 mbar), plasma ashing (> 24 hours, cool plasma conditions), and microwave (wet) digestion (H2SO4 (96%); max. 500 W). The test items retained in lung tissue were determined using ion-coupled plasma mass spectroscopy (ICP-MS) using the prepared samples after recommended dilution with deionized water.
Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on the method of lung burden analysis.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- 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
HISTOPATHOLOGY: Yes
- 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 1 (Cobalt Zinc Aluminate Blue Spinel) high dose group after 90 days nose-only inhalation: adrenals, aorta, bone marrow (and/or fresh aspirate), brain (including sections of cerebrum, cerebellum, and medulla/pons), caecum, coagulating glands, colon, duodenum, epididymides, femur and stifle joint, heart, ileum, jejunum, kidneys, larynx (3 levels incl. the base of the epiglottis), liver, lung (left lobe at three levels, including main bronchi), lymph nodes from the hilar region of the lung, lymph nodes (distal from the portal-of-entry), mammary gland (males and females), muscle (thigh), nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT)), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, peripheral nerve (sciatic or tibial), pituitary, prostate, rectum, salivary glands, seminal vesicles, skin, spinal cord (cervical- mid-thoracic, and lumbar), spleen, sternum, stomach, teeth, testes, thymus, thyroid, trachea (at least two levels incl. 1 longitudinal section through the carina and 1 transverse section), urinary bladder, uterus, vagina, all gross lesions and masses.
The following respiratory tract organs of animals 101-110 and 201-210 of group 1 and 4, 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 will be performed in 10 animals per sex of the control and high dose group at day 1 post-exposure.
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

Results of examinations

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:
no effects observed
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:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Lungs showed typical dose-dependent discolourations caused by the test item:
- 1 day post exposure: 1/20 males and 1/10 females of the control group, 3/20 males and 1/10 females of the low dose group, 5/20 males and 4/10 females of the mid dose group, and 18/20 males and 10/10 females of the high dose group
- 90 days post exposure: 1/5 males and 0/5 females of the controls, 0/5 males and 3/5 females of the low dose group, 2/5 males and 1/5 females of the mid dose group, and 4/5 males and 5/5 females of the high dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
High dose group:
- In the lung, exposure-related findings represent very slight multifocal granulocytic cell infiltration in the alveoli in 6/10 male and 5/10 female rats. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (7 slight; 3 moderate) and 10/10 females (8 slight; 2 moderate) as well as in the bronchus-associated lymphoid tissue in 6/10 males (all very slight) and in 7/10 females (all very slight). This lesion correlated with a macroscopically observed multifocal discoloration of up to 1 mm in diameter in the lung. A multifocal very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization) was seen in 1/10 males.
- In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 3/10 males and 3/10 females in the nose-associated lymphoid tissue (NALT). Further, very slight multifocal hyaline droplets were seen in the nasal epithelium in 1/10 male and 2/10 female rats.
- In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (7 very slight; 3 slight) and in 7/10 (3 very slight; 4 slight) females. This lesion correlated with a macroscopically observed enlargement in few animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- At day-1 after the last exposure, females of the low and high dose groups showed statistically significantly increased absolute (+26.3% and +29.9%, respectively) and relative (+23.4% and +26.8%) weights of the left adrenal, when compared to the vehicle control group. However, no such effects were observed for the right adrenals. Moreover, the effect was transient, no effects on adrenal weights were observed in any male dose group, and the finding in females was without a histopathological correlate. Thus, the findings are considered to be not biologically relevant.
- At day-1 after the last exposure, females of the high dose group showed marginally but statistically significantly increased absolute and relative weights of the left kidney (+10.5% and 8.4%, respectively), when compared to the vehicle control group. However, no such effects were observed for the right kidneys. Moreover, the effect was transient, no effects on kidney weights were observed in any male dose group, and the finding in females was without a histopathological correlate. Thus, the findings are considered to be not biologically relevant.

GROSS PATHOLOGICAL FINDINGS:
- Enlarged lung-associated lymph nodes (LALN), as a particle-specific lung clearance pathway, were observed only incidentally (1 day post exposure: 2/20 males and 1/10 females of the high dose group; 90 days post exposure: 1/5 females of the control and low dose group).
- Some other incidental macroscopic observations in other organs were obtained (1 day post exposure: 1/20 males of the low dose group, 1/20 males and 1/10 females of the mid dose group, and 2/20 males and 1/10 females of the high dose group; 90 days post exposure: 1/5 females of the control group, 1/5 females of the mid dose group, and 1/5 females of the high dose group).

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.

HAEMATOLOGICAL FINDINGS:
- On day 1 post exposure, the male low dose group showed a statistically significant decrease of the red blood cell count (-4.4%), when compared to the vehicle control group. Moreover, the haemoglobin concentration was statistically significantly decrease in the mid dose group (-3.1%). The haematocrit value was statistically significantly decreased in the low and mid dose group (-3.6% and -3.7%, respectively). Furthermore, the differential cell count revealed a statistically significant increase (+46.2%) in the proportion of banded neutrophils in the male low dose group, when compared to the vehicle control group. These effects were marginal, not dose-related, and not observed in females. Thus, the findings are considered to be not biologically relevant.

BALF ANALYSES:
- At days 1 and 90 (females only) post-exposure no statistically significant increases of polymorphonuclear neutrophils were detected in any treatment group. The PMN percentages (in the range from 1.2% to 3.3%, males - 1.3% to 2.0%, females) are close to historical clean air control data; thus, the test item did not induce a relevant PMN-related lung inflammation. Lymphocyte levels were also found low and at control levels (<1%).
- For lactic dehydrogenase, ß-glucuronidase and total protein, no relevant statistically significant increases were detected in any group of both sexes and sacrifice dates.

LUNG BURDEN ANALYSIS
- Lung weight: The lung burden analysis (males only) did not show any statistically significantly increase in the lung to bodyweight ratio at post-exposure day-1, 28, and 92, when compared to the vehicle control group.
- Chemical analysis: Information will be added to the robust study summary upon availability
Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on results of the lung burden analysis.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
6 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: observed at lung overload conditions, in exceedance of the maximum tolerated dose (MTD)

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.4, 1.5 and 6 mg/m³ of Cobalt Zinc Aluminate Blue Spinel.

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, clinical chemistry, lung weights, bronchoalveolar lavage fluid (BALF) analysis, histopathological evaluation.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 1 - Cobalt Zinc Aluminate Blue Spinel) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages. Very slight multifocal hyaline droplets were seen in the nasal epithelium. These lesions are considered to represent a non-adverse adaptive change. Moreover, very slight multifocal granulocytic cell infiltration in the alveoli were observed. A single male showed very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization).

Under the conditions of this test, based on very slight granulocytic cell infiltration in the lung, a NOAEC of 6 mg/m³ was derived for both sexes, being the maximum tolerated concentration based on the more than 2-fold increase lung clearance half-time (further details are reported in the study record in the toxicokinetics section). Consequently, the pigment is to be regarded as inert material when tested up to the maximum tolerated concentration as defined by significant impairment of lung clearance (Driscoll and Borm, 2020)*.

*References:
- Driscoll, K.E. and Borm, P.J.A., 2020. Expert workshop on the hazards and risks of poorly soluble low toxicity particles. Inhal Toxicol 32(2):53-62.