Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start: 2020-07-30 and still ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

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 only 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:
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 iron oxide
Test material form:
solid: particulate/powder
Details on test material:
- OLD Chemical description: Chromium iron oxide
- NEW Chemical description: Chromium iron hematite
- Substance type: inorganic pigment
- State of aggregation: solid, black powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature

Test animals

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

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet: 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

IN-LIFE DATES: From: 2020-08-04 To: 2021-02-17

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 / concentrationsopen allclose all
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:
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

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 (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 (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
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 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 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:
not specified
Description (incidence and severity):
Information will be added to the robust study summary upon availability
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.

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:
- The lung burden analysis did not show any statistically significantly increase in the lung to bodyweight ratio at post-exposure day-1 and 92, when compared to the vehicle control group.
- The results of the chemical lung burden analysis will be submitted later.

Effect levels

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

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 10mg/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, histopathological evaluation.
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 NOAEC is considered to be above 10 mg/m³, the maximum tolerated concentration based on lung burden calculations.


The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct. Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:
(i) chemical analysis of lung burden
(ii) clinical chemistry
(iii) QAU evaluation and study finalisation