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Repeated dose toxicity: inhalation

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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
Remarks:
ongoing 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:
Manganese alumina pink corundum
EC Number:
269-061-0
EC Name:
Manganese alumina pink corundum
Cas Number:
68186-99-2
Molecular formula:
Mn(x)Al(2-x)O3 0,01≤x≤0,25
IUPAC Name:
Manganese aluminium corundum
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Manganese Alumina Pink Corundum
- Substance type: inorganic pigment
- Physical state: solid, pink 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: 2021-09-15

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.58 - <= 1.88 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: mass median aerodynamic diameter (MMAD) was determined 2-3 times using 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 Manganese Alumina Pink Corundum 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: ± 0.05 mg/m³; 0.03 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
1.51 mg/m³ air (analytical)
Remarks:
SD: ± 0.18 mg/m³; 0.125 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
6 mg/m³ air (analytical)
Remarks:
SD: ± 0.37 mg/m³; 1.0 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

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 4.5% (rel. density=4, 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 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 (determination of lung:body weight ratio before and after treatment as well as chemical analysis)
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
- Chemical analysis: After sacrifice the lungs were prepared by freeze drying (> 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.
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 2 (powder Manganese Alumina Pink Corundum) 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 7, respectively, were examined histopathologically: Nasal and Paranasal Cavities, pharynx (Laryngopharynx/Nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology 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:
effects observed, treatment-related
Description (incidence and severity):
High dose groups:
On post exposure day 1, in males the absolute and in females the relative lung weights were statistically significantly increased (+9.5% and +6.6%, respectively) as compared to concurrent controls; because of the small increases in absolute figures and the normalisation after 3 months of recovery these findings are considered as not adverse.
Gross pathological findings:
no effects observed
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, exposure-related findings represent very slight multifocal granulocytic cell infiltration in the alveoli in 8/10 male and 10/10 female rats. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (7 slight; 3 moderate) and 10/10 females (5 slight; 5 moderate) as well as in the bronchus-associated lymphoid tissue in 9/10 males (all very slight) and in 10/10 females (all very slight). This lesion correlated with a macroscopically observed multifocal discoloration of up to 1 mm in diameter in the lung. Further in the lung interstitium, there was a multifocal mononuclear cell infiltration in 5/10 males (all very slight) and in 4/10 females (all very slight). This mononuclear cell infiltration was mainly observed in the vicinity of the terminal bronchi. A multifocal very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization) was seen in 1/10 males and 3/10 females.
- In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 9/10 males and 8/10 females in the nose-associated lymphoid tissue (NALT).
In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 6/10 males (3 very slight; 3 slight) and in 9/10 (6 very slight; 3 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:
MORTALITY:
- Two rats, one male (#5123, low dose group) and one female (#6213, mid dose group) rat dropped out and had to be killed in moribund condition. The male showed loss in body weight and a reddish pancreas, and the female suffered from a node on the back.

WATER CONSUMPTION:
- Some observed statistically significantly increased values were observed in the low dose females from post exposure day 106 to 176. These altered values were considered as incidental findings as these effects were without a clear dose-dependency.

HAEMATOLOGICAL FINDINGS:
- One day after the last exposure, mid dose males showed a marginal but statistically significant decrease (3%) in the haemoglobin concentration, when compared to the clean air control. This finding is considered to be of an incidental nature since no such effect was observed in the high dose group or in any female dose group.

CLINICAL BIOCHEMISTRY FINDINGS:
- One day after the last exposure, high dose females showed a slight but statistically significant increase (+12.2%) in the creatinine concentration, when compared to the clean air control. No such effect was observed in any female dose group.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
- On post exposure day 1, females of the mid dose group showed a statistically significantly increased absolute and relative left adrenal weight (+34.3% and +34.9%, respectively), when compared to the clean air control group. No such effect was observed in the high dose group or any male dose group. On post exposure day 92, the values were similar to the control levels. The finding was without a histopathological correlate. Thus, the finding is considered to be not biologically relevant.
- On post exposure day 1, females of the low, mid, and high dose group showed a statistically significant increase in the relative weight of the left kidney (+8.8%, +10.9%, and +10.9%, respectively), when compared to the clear air control. The right kidney relative weight was statistically significantly altered (+8.7%) only in mid-dose females. On postexposure day 92, females of the high dose group showed a statistically significantly increased relative weight of the left and right kidney (+11.2% and +17.9%, respectively). No such effects were observed in any male dose group. Moreover, the finding was without a histopathological correlate. Thus, the finding is considered to be not biologically relevant.
- On post exposure day 92, males of the low and mid dose groups showed a statistically significantly decrease in the absolute brain weight (-6.9% and -5.5%, respectively), when compared to the clear air control group. However, no such effect was observed in the high dose group or any female dose group. The relative weights were not statistically significantly changed. Moreover, the finding was without a histopathological correlate. Thus, the finding is considered to be not biologically relevant.

GROSS PATHOLOGICAL FINDINGS:
- Only incidental macroscopic observations were obtained: The LALN showed enlargement in 1/5 females of the control group (only post exposure day 90), in 2/20 males of the low dose group (only post exposure day 1), 1/9 and 1/4 females of the mid dose group (post exposure day 1 and 90, respectively), and 1/20 males and 1/10 females of the high dose group (only post exposure day 1). The lung showed discoloured areas in 1/20 males and 1/10 females of the control group on post exposure day 1 and 1/5 males of the control group on post exposure day 90; In 5/20 males and 1/10 females of the low dose group on post exposure day 1 and 1/4 males and 1/5 females of the low dose group on post exposure day 90; In 1/20 males and 4/10 females of the mid dose group on post exposure day 1 and 1/5 males and 1/4 females of the mid dose group on post exposure day 90; in 1/20 males and 2/10 females of the high dose group on post exposure day and 1/5 males of the high dose group on post exposure day 1. Other organs showed incidental findings in 1/5 control females on post exposure day 90, 2/20 males of the low dose group on post exposure day 1 and 1/5 males on post exposure day 90, 1/9 and 1/4 females of the mid dose group on post exposure day 1 and 90, and 1/10 females of the high dose group on post exposure day 1 and 1/5 males on post exposure day 90.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- In the clean air control group, single lesions were seen in different organs, which represented commonly found background lesions in this rat strain.
- All the other findings within the different organs occurred in single animals of the high dose group and were interpreted to be incidental without any relation to the exposure.


LUNG BURDEN ANALYSIS:
The clearance half-times of the test item amounted to 51 and 45 days in the low and mid dose group, respectively, thus were very close to the physiological half-time of approx. 50-60 days . In the high dose group, a half-time of 105 days was determined; this is an approx. 2-fold increase as compared to the physiological value. This finding in the high dose group is indicative for an overload of particle clearance condition (Driscoll and Borm, 2020).

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
other: BALF
Remarks on result:
other: the highest concentration resulted in a more than 2-fold increase in lung clearnace half-time, indicating an overload of particle clearance condition, i.e. defining the maximum tolerated concentration

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 Manganese alumina pink corundum.
All animals survived (but two due to incidental effects) 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, bronchoalveolar lavage fluid (BALF) analysis.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 2 - Manganese alumina pink corundum) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages. These lesions are considered to represent a non-adverse adaptive change. Moreover, alveolar granulocytic cell infiltration and interstitial mononuclear cell infiltration was observed in the lung. A few animals showing 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 at the maximum tolerated concentration based on lung burden calculations. In the high dose group, a half-time of 105 days was determined; this is an approx. 2-fold increase as compared to the physiological value. This finding in the high dose group is indicative for an overload of particle clearance condition (Driscoll and Borm, 2020). 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)*.


The measures implemented during the ongoing Covid-19 pandemic led to a delay in laboratory capacities and delays in the study conduct (see also the attached documentation under "Attached justification"). Due to this the following parameters are not yet available and will be added to the robust study summary upon availability:
(i) QAU evaluation and study finalisation

*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.