Manganese alumina pink corundum

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-04 to 2021-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

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 manganese alumina pink corundum at doses of 0.15, 0.5 or 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:

0.15, 0.5 and 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).

*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.

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 (up to 2 mm in diameter) in the pigment powder treated groups. In the low-dose group, LALN were slightly enlarged and some light areas on lung (Ø < 1 mm) in one animal; others in this group were without any special findings. In the mid-dose group, LALN were slightly to moderately enlarged in the animals, and in the lung of one animal, some light areas (Ø 2 mm) were observed. Also in the high-dose group, the LALN were moderately enlarged in two animals, and there some light areas (Ø 2 mm) observed, while the rest of the animals in this group were without any special findings.

Other findings:

BRONCHOALVEOLAR LAVAGE FLUID ANALYSIS:
At day 3 post-treatment, statistically significant and treatment-related alterations in the percentage of polymorphnuclear neutrophils (PMN: 10.06 ± 9.04 %; P<0.1 %) were observed in the high-dose treated group compared to the controls (1.04 ± 1.25 %). Also in the high-dose group, the percentage of macrophages was statistically significant decreased (89.25 ± 9.13 %) compared to the controls (98.99 ± 1.41 %). The low- and mid-dose animals showed %PMN (1.25 ± 0.54 % and 1.44 ± 0.66 %) and %macrophage values (98.44 ± 0.72 % and 97.88 ± 1.05 %) in the range of the control control group.

Conclusions:

Female Wistar rats were exposed to manganese alumina pink corundum at doses of 0.15, 0.5 or 2 mg by intratracheal instillation (total dose was instilled on two consecutive days) to investigated 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 all pigment powder treated groups.
The bronchoalveolar fluid analysis showed a statistically significant decrease in the percentages of macrophages (89 %) and an increase of PMNs (10 %) in the high-dose group compared to the controls (99 % and 1 %). The low- and mid-dose animals showed %PMN (1.25 % and 1.44 %) and %macrophage values (98 % and 98 %) in the range of the control control group.

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).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-04 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

2018-06-25

Deviations:

yes

Remarks:

Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional in guideline)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2018-11-22

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light.

Species:

rat

Strain:

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 (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

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 ± 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

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). 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

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

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day (with the exception of weekends and public holidays: once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: 1 day before treatment and twice a week in the first 4 and once a week thereafter throughout the study for all animals

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was determined for each group on a weekly basis.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: YES
- Water consumption was determined for each group on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: red blood cells (RBC), haemoglobin (HB), haematocrit (HCT), reticulocytes (RET), mean cell volume (MCV), mean haemoglobin/erythrocyte (MCH), mean haemoglobin concentration/erythrocyte (MCHC), prothrombin time (PT), thromboplastin time (TP), total white blood cells (WBC), differential white cell count (% and absolute), platelets (PTL)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), urea, triglycerides, total bilirubin, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB, glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K), phosphorous (P), chloride (CL)

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 90 days post-exposure
- Dose groups that were examined: all
- Number of animals: 5 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.
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: YES
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 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.

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). Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on results of the lung burden analysis.

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

Critical effects observed:

no

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 the approx. 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)*.

*Reference:
- 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Objective of study:

absorption

other: lung clearance

Test guideline

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.

Test material

Test material information

Constituent 1

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

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

Test animals

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 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

Administration / exposure

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: 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

Duration and frequency of treatment / exposure:

13 weeks (65 exposure days), 6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

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)

Doses / concentrations 2

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)

Doses / concentrations 3

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 / 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 4.5% (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

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 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.

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): 55Mn;
Internal standards (m/z): Chromium: 45Sc, 74Ge.
Limit of quantification: < 1 µg/L Mn

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

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.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Lung burden with manganese alumina pink corundum after 1, 28, and 90 days after the 90-day exposure period:
- control: < LOQ;
- 0.4 mg/m3: 0.08, 0.05 and 0.02 mg/lung;
- 1.51 mg/m3: 0.32, 0.21 and 0.08 mg/lung;
- 6 mg/m3: 1.97, 1.7 and 1.07 mg/lung.

Main ADME results 2

Type:

other: lung clearance half-time

Results:

after exposure to 0.4 mg manganese alumina pink corundum/m3: 51.3 days

Main ADME results 3

Type:

other: lung clearance half-time

Results:

after exposure to 1.51 mg manganese alumina pink corundum/m3: 44.7 days

Main ADME results 4

Type:

other: lung clearance half-time

Results:

after exposure to 6 mg manganese alumina pink corundum/m3: 103.4 days

Toxicokinetic / pharmacokinetic studies

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

Metabolite characterisation studies

Metabolites identified:

not measured

Enzymatic activity

Enzymatic activity measured:

not examined

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

not examined

Applicant's summary and conclusion

Conclusions:

Male rats were exposed to concentrations of 0.4, 1.51 and 6 mg manganese alumina pink corundum/m3 for 6 hours per day, 5 days/week for 90 days via nose-only inhalation. The lung burden and lung clearance with manganese alumina pink corundum 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 0.09, 0.05 and 0.02 mg/lung, in the mid-dose groups 0.33, 0.21 and 0.08 mg/lung, and in the high-dose groups 1.97, 1.70 and 1.07 mg/lung of the test item Manganese Alumina Pink Corundum (Pigment 2) were determined, respectively. 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. 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 105 days was determined, being above approx. 2-fold increase as compared to the physiological values of 50.5 or 60 days. The 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).