Chromium iron oxide

Repeated dose toxicity: inhalation

Administrative data

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

Cross-reference

Cross-reference 1

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:

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 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:

Crl:WI (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. 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 (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 (°C): 22 ± 2
- Humidity(%): 55 ± 15
- Photoperiod (hrs dark/light): 12 / 12

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.
- 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.:

0.6 mg/m³ air (analytical)

Remarks:

SD: ± 0.09 mg/m³; 0.1 mg/lung (calculated total dose using MPPD v3.04)

Dose / conc.:

2.5 mg/m³ air (analytical)

Remarks:

SD: ± 0.35 mg/m³; 0.4 mg/lung (calculated total dose using MPPD v3.04)

Dose / conc.:

10.02 mg/m³ air (analytical)

Remarks:

SD: ± 1.49 mg/m³; 1.7 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); 5 males (90 days recovery)

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: 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 0.6, 2.5 and 10 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 4.7% (rel. density=5.1, 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 0.6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.6 mg/m³ x 4.7% = 0.13 mg/lung
Example for deposited mass at 2.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2.5 mg/m³ x 4.7% = 0.55 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 (1 mL) and HNO3 (4 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 analyzed by ICP-MS. In the case of HF having been added during the digest 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.

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 (1 mL H2SO4 (96%) and 4 mL HNO3; 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 with deionised water samples were analyzed by ICP-MS. In the case of HF having been added during the digest H3BO3 was added during sample dilution. Quantification was achieved against matrix matched standards. To ensure validity of the analysis data, samples were bracketed by QC standards.

Parameter/Setting:
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): 52Cr; 53Cr;
Internal standards (m/z): Chromium: 45Sc; 74Ge.
Limit of quantification: 0.1 ng Cr/mL dilution

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 in IUCLID section 7.2.4.

Type:

absorption

Results:

Lung burden with chromium iron oxide after 1, 28, and 90 days after the 90-day exposure period:
- 0.6 mg/m3: 141, 89 and 39 µg/lung;
- 2.5 mg/m3: 531, 341 and 225 µg/lung;
- 10.02 mg/m3: 1924, 1972 and 895 µg/lung.

Type:

other: lung clearance half-time

Results:

after exposure to 0.6 mg chromium iron oxide/m3 air: 49.5 days

Type:

other: lung clearance half-time

Results:

after exposure to 2.5 mg chromium iron oxide/m3 air: 81.5 days

Type:

other: lung clearance half-time

Results:

after exposure to 10.02 mg chromium iron oxide/m3 air: 80.6 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 measured

Details on excretion:

not measured

Metabolites identified:

not measured

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability) testing results:

not measured

Conclusions:

Male rats were exposed to concentrations of 0.6, 2.5 and 10.02 mg chromium iron oxide/m3 air for 6 hours per day, 5 days/week for 90 days via nose-only inhalation. The lung burden and clearance with chromium iron oxide were determined 1, 28 and 90 days after the 90-day exposure period. A vehicle control group was run concurrently.

One day, 1 month and 3 months after end of exposure, in the low-dose groups 0.14, 0.09 and 0.04 mg/lung, in the mid-dose groups 0.53, 0.34 and 0.23 mg/lung, and in the high-dose groups 1.92, 1.97 and 0.90 mg/lung of the test item chromium iron oxide (Pigment 3) were determined, respectively. The retained masses correspond quite well to the theoretical data derived from the MMPD model prediction.
During the recovery period a retarded lung clearance was observed in the high-dose group. In contrary, in the mid- and low-dose groups a partial and a physiological lung clearance was found, respectively. This reflects well the different grade of clearance retardation due to the various lung loads.
The clearance half-times of the low concentration groups with 50 days was in the range to the physiological half-time of approx. 60 days (ECETOC, 2013), or 50.5 days (median over all 5 sub-chronic inhalation toxicity studies, low-dose animals). In the mid- and high-dose group, a half-time of 81 days was determined, being above a 1.5-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 (Driscoll and Borm, 2020). Although the 2-fold increase in lung clearance half-time retardation has not been reached, it is assumed that chromium iron oxide still fulfils the criteria as being a PSLT and that additional concentration groups with higher exposure would not have yielded any other effects than local inflammation at presence of higher clearance-half time values.

Cross-reference 2

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:

Female Wistar rats were exposed to chromium iron oxide at doses of 0.2, 0.8 or 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 this study 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 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).

*Reference:
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. The lung associated lymph nodes (LALN) were moderately enlarged in one animal of the mid-dose group, and slightly to moderately enlarged in two animals of the high-dose group. There were some dark grey areas on whole lung in all animals of the mid- and high-dose group.The animals in the low-dose group, were without any special findings.

Other findings:

ORGAN WEIGHT:
The absolute lung weight was statistically significant increased in the low- and high-dose group (1.065 ± 0.028 g and 1.070 ± 0.046; P< 0.05) 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, a statistically significant increase in the percentage of lymphocytes were observed in the animals of the high-dose group (1.31 ± 0.55 %; P < 0.05) compared to the controls (0.08 ± 0.20 %). The low- and mid-dose animals showed %lymphocyte values in the range of the control animals (1.13 ± 1.27 and 0.31 ± 0.38 %).

Conclusions:

Female Wistar rats were exposed to chromium iron oxide at doses of 0.2, 0.8 or 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), and some dark grey areas on whole lung in all animals of the mid- and high-dose group. The absolute but not the relative lung weights were increased in the animals of the low- and high-dose groups compared to controls.

At day 3 post-treatment, a statistically significant increase in the percentage of lymphocytes were observed in the animals of the high-dose group (1.31 %; P < 0.05) compared to the controls (0.08 %). The low- and mid-dose animals showed %lymphocyte values in the range of the control animals (1.13 and 0.31%).

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

Test material information

Constituent 1

Reference substance name:

Chromium iron oxide

EC Number:

235-790-8

EC Name:

Chromium iron oxide

Cas Number:

12737-27-8

Molecular formula:

Fe(x)Cr(2-x)O3 0,65≤x≤1,75

IUPAC Name:

chromium(3+) iron(3+) trioxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Chromium iron oxide
- Substance type: inorganic pigment
- State of aggregation: solid, black powder, odourless, Hematite-corundum structure
- 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.

Test animals

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

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.48 - <= 2.05 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.
- 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 0.6, 2.5 and 10 mg/m³ Chromium Iron Oxide 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 / concentrations

Doses / concentrations 1

Dose / conc.:

0.6 mg/m³ air (analytical)

Remarks:

SD: ± 0.09 mg/m³; 0.1 mg/lung (calculated total dose using MPPD v3.04)

Doses / concentrations 2

Dose / conc.:

2.5 mg/m³ air (analytical)

Remarks:

SD: ± 0.35 mg/m³; 0.4 mg/lung (calculated total dose using MPPD v3.04)

Doses / concentrations 3

Dose / conc.:

10.02 mg/m³ air (analytical)

Remarks:

SD: ± 1.49 mg/m³; 1.7 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). For detailed information on the preliminary dose-range finding study (DRF A) please refer to the study record "s_Creutzenberg_2022" in the IUCLID sction 7.2.4.
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.6, 2.5 and 10 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 4.7% (rel. density=5.1, 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 0.6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.6 mg/m³ x 4.7% = 0.13 mg/lung
Example for deposited mass at 2.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2.5 mg/m³ x 4.7% = 0.55 mg/lung
Example for deposited mass at 10 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 10 mg/m³ x 4.7% = 2.2 mg/lung
Retained mass at 10 mg/m³: approx. 1.7 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: 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.

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 females (90 days post-exposure) and 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 "k_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 WEIGHT: Yes
organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart. The absolute and relative lung weights were calculated.

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 3 (Chromium iron oxide) high dose group after 90 days nose-only inhalation: 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 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 was performed in 10 animals per sex of the control and high dose group at day 1 post-exposure.

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.

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):

- One day after the last exposure, enlarged lung-associated lymph nodes (LALN) were observed in 5 out of 20 male rats of the high dose group (vs. 1/20 in controls). This is a particle-specific lung clearance pathway.
- Lungs showed typical dose-dependent discolorations caused by the test item. On post-exposure day 1, the incidence for males was 0/20, 8/20, 18/20, 20/20 in the control, low, mid, and high dose group, respectively. On post-exposure day 90, the incidence for males was 0/5, 1/5, 5/5, 5/5 in the control, low, mid, and high dose group, respectively. On post-exposure day 1, the incidence for females was 1/10, 2/10, 9/10, 10/10 in the control, low, mid, and high dose group, respectively. On post-exposure day 90, the incidence for females was 0/5, 4/5, 4/4, 5/5 in the control, low, mid, and high dose group, respectively.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- As exposure-related finding only an accumulation of particle-laden macrophages was observed in different parts of the respiratory tract.
- In the lung, a multifocal accumulation of particle-laden macrophages was seen in the alveoli in all male (all slight) and female (all slight) animals as well as in the bronchus-associated lymphoid tissue (BALT) in 9/10 males (8 very slight and 1 slight) and in all females (9 very slight and 1 slight). This lesion correlated with a macroscopically observed dark grey multifocal discoloration of up to 1mm in diameter in the lung.
- 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).
- In the lung-associated lymph nodes (LALN), there was a multifocal very slight accumulation of particle-laden macrophages in 7/10 males and in 5/10 females. This lesion correlated with a macroscopically observed enlargement in few animals.
- The accumulation of particle-laden macrophages was not associated with further pathologic lesions in any organ.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

MORTALITY:
- Due to a trauma to the tail, one animal (female # 3202; mid dose group) was sacrificed preterminally. The trauma was diagnosed as an acute fracture of the tail. The lesion was interpreted to be incidental without any relation to the exposure.

CLINICAL BIOCHEMISTRY FINDINGS:
- In males, the alanine aminotransferase activity was statistically significantly decreased by 21.1% and 7.2% at concentration levels of 0.6 and 2 mg/m³, respectively, on post-exposure day 1 (please refer to: ‘Attached background material’). Moreover, the urea concentration was statistically significantly decreased (-17.3%) in low-dose males. However, these effects were not observed either in higher dose groups or in females. Thus, these effects are considered to be incidental and without toxicological relevance.
- In females exposed to 0.6 mg/m³, the chloride level was statistically significantly increased on post-exposure day 1 (please refer to: ‘Attached background material’). No such effect was observed either at higher concentrations or in males. Thus, this effect is considered to be incidental and without toxicological relevance.

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- At day-92 after the last exposure, females of the high dose group showed statistically significantly increased absolute and relative weights of the left (+39% and +39.9%, respectively) and right ovary (+36.5% and +37.2%, respectively), when compared to the vehicle control group. The effect showed, however, no clear dose-response relationship and was without a histopathological correlate. Thus, the finding is considered to be not biologically relevant.
- At day 92 after the last exposure, males of the high dose group showed a statistically significantly increased relative thymus weight (+42.2%). The absolute weight was, however, not statistically significantly altered. Moreover, no such effect was observed in females and the finding was without a histopathological correlate, and thus, considered to be not biologically relevant.

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.

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 2.9% to 5.9%, males - 0.7% to 1.9%, 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 weights: In both sexes, no statistically significant changes as compared to concurrent controls.

LUNG BURDEN ANALYSIS:
Please refer to IUCLID section 7.1.1 "k_Creutzenberg_2022_lung burden" for detailed information on results of lung burden analysis.

Effect levels

Key result

Dose descriptor:

NOEC

Effect level:

10.02 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no overt toxicity seen, when tested up to the maximum tolerated concentration as defined by significant impairment of lung clearance

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 0.6, 2.5 and 10 mg/m³ of chromium iron oxide.
All animals (but one due to accident) 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, lung weights, bronchoalveolar lavage fluid (BALF) analysis.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 3 - Chromium iron oxide) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages, which was not associated with further pathologic lesions in any organ. The lesion occurred statistically significant in the lung and in the lung-associated lymph nodes. This lesion is considered to represent a non-adverse adaptive change.

Due to an absence of any adverse findings the NOEC is considered to be 10 mg/m³, the maximum tolerated concentration based on lung burden calculations and the overall negative toxicological profile, showin no concern for local or systemic toxicity.

The chemical lung burden analysis revealed that the estimated lung burdens on day 90 using the MPPD (v3.04) model were met – the actual lung burden on day 93 were in good agreement with the estimated values. The exponential regression analysis of the lung burdens over days 3, 28 and 90 post-exposure calculated clearance half-times of 49.5, 81.5, and 80.6 days in rats of the low, intermediate, and high dose group. Further evaluation of the lung clearance may involve an (i) interval-specific estimation of the clearance half-times to illustrate the early and late response of the lung clearance and (ii) an estimation of the alveolar macrophage loading to further clarify the level of lung clearance impairment.
However, the available data and first tier evaluation already clearly shows that the increase of the clearance half-times above the physiological values of approx. 50 days (± 5 days) are indicative for beginning impairment of lung clearance, which is a key event in the development of lung overload (Driscoll and Borm, 2020). Consequently, the study design of this sub-chronic inhalation toxicity study fulfilled the criteria of having tested up to the maximum tolerated concentration, which is either limited by toxicity or testing up to concentrations which are indicative for pre-lung overload but not reaching clear lung overload. It appears highly unlikely that the testing at concentrations above the high-concentration used in this study would lead to any other conclusion than an absence of test-item specific toxicity.