Zirconium praseodymium yellow zircon

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 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 zirconium praseodymium yellow zircon 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 DRF A were conducted under non-GLP conditions because of the screening, dose-range finding nature of the experiments. However, the experimental procedures followed the GLP rules (e.g. use of SOPs and documentation/archiving).

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 8 weeks old
- Weight at study initiation: approx. 182 g
- Housing: housed in Makrolon® (polycarbonate) cages type Ill, four rats per cage in the treated groups and three rats per cage in the vehicle control group; absorbing softwood ('ssn BK 8-15') bedding material.
- Diet: commercial chow in pellet form (ssniff”V1534; supplier: ssniff Spezialdiäten GmbH, Soest, Germany)
- Water: tap water
- Acclimation: approximately one week the animals were allowed to adjust and become acclimatized to the Fraunhofer ITEM environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: intratracheal instillation

Vehicle:

other: saline solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in vehicle, each aliquot in a volume of 0.3 mL. After gentle shaking all samples were sonicated for 2 minutes to guarantee a homogeneous suspension. After sonication samples were shaken again to perpetuate the homogeneity until administration to the animals.
The total dose was instilled in two aliquots on two consecutive days to achieve a homogeneous distribution of the test material in lungs.

Doses:

0.2, 0.8 and 3.2 mg

No. of animals per sex per dose:

4 females in 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-related macroscopical findings were observed, like enlarged lung associated lymph nodes (LALN) or white areas in some pigment powder treated groups. In the low dose group, LALN were moderately enlarged but the lungs were without any special findings. The LALN of two animals in the mid-dose group were slightly enlarged, and the lungs of two other animals of this group showed some white areas. Two animals of the high dose group showed some white areas in the lung, and one of these animals had moderately enlarged LALN.

Other findings:

ORGAN WEIGHT:
The absolute lung weight was statistically significant increased in the low- and high-dose groups (1.084 ± 0.043 g and 1.100 ± 0.048 g; P< 0.05 or 0.01) compared to the controls (0.985 ± 0.051 g), but the relative lung weight was not increased.

BRONCHOALVEOLAR LAVAGE FLUID ANALYSIS:
At day 3 post-treatment, statistically significant decrease in the percentage of macrophages (92.94 ± 2.66 %; P<0.05 %) compared to the controls (98.99 ± 1.41 %). The low- and mid-dose animals showed %macrophage values in the range of the control animals (98.94 ± 0.24 % and 97.00 ± 1.02 %).

Conclusions:

Female Wistar rats were exposed to zirconium praseodymium yellow zircon 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.

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-related macroscopical findings were observed, like enlarged lung associated lymph nodes (LALN) or white areas in some pigment powder treated groups. The absolute but not the relative lung weights were increased in the animals of the low- and high-dose groups compared to controls.
The bronchoalveolar fluid analysis showed a statistically significant decrease in the percentage of macrophages (93 %) compared to the controls (99 %) at day 3 post-treatment. The low- and mid-dose animals showed %macrophage values in the range of the control animals (99 % and 97 %).

On the basis of the results obtained in the BALF analysis after in vivo instillation of five different inorganic pigments combined with deposition modelling (using the MPPD model), the pigment with the highest reactivity in the respiratory tract was used for a subsequent 14-day inhalation dose-range finding study.
For the other four inorganic pigments, the concentrations for the main 90-day study was based on the results obtained in this instillation experiments combined with deposition modelling (using the MPPD model).

Cross-reference 2

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:

2020-08-0 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:

Wistar (Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in polycarbonate Makrolon 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 (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs 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 three to 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: ± 0.08 mg/m³; 0.1 mg/lung (calculated total dose using MPPD v3.04)

Dose / conc.:

2.5 mg/m³ air

Remarks:

SD: ± 0.38 mg/m³; 0.4 mg/lung (calculated total dose using MPPD v3.04)

Dose / conc.:

9.99 mg/m³ air (analytical)

Remarks:

SD: ± 0.94 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), 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

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

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). Samples were transferred into Pt-crucible and underwent high-temperature fusion using Na2CO3 and Na2B4O7. The melt was taken up in 5 N HCl, with the final sample volume being 50 mL. After appropriate dilution (see raw data) with deionised water samples were analysed by ICP-MS. Since hydrofluoric acid (HF) was used for 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.

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): 141Pr;
Internal standards (m/z): Chromium: 115In, 175Lu.
Limit of quantification: 0.25 µg Pr/lung

Statistics:

Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.

Preliminary studies:

A dose range finding study by intratracheal instillation was conducted. For further information please refer to the study record in section 7.2.4.

Type:

absorption

Results:

Lung burden with zirconium praseodymium yellow zircon after 1, 28, and 90 days after the 90-day exposure period:
- control: < LOQ;
- 0.6 mg/m3: 252, 149 and 84 µg/lung;
- 2.5 mg/m3: 1475, 1280 and 1161 µg/lung;
- 9.99 mg/m3: 5247, 4760 and 3868 µg/lung.

Type:

other: lung clearance half-time

Results:

after exposure to 0.6 mg zriconium praseodymium yellow zircon: 59.2 days

Type:

other: lung clearance half-time

Results:

after exposure to 2.5 mg zriconium praseodymium yellow zircon: 277.2 days

Type:

other: lung clearance half-time

Results:

after exposure to 9.99 mg zriconium praseodymium yellow zircon/m3: 210.0 days

Details on absorption:

For detailed information of absorption in lung tissue please refer to the filed "overall remarks, attachments".

Details on distribution in tissues:

not examined

Details on excretion:

not examined

Metabolites identified:

not measured

Enzymatic activity measured:

not examined

Bioaccessibility (or Bioavailability) testing results:

not examined

Conclusions:

Male rats were exposed to concentrations of 0.6, 2.5 and 9.99 mg zirconium praseodymium yellow zircon/m3 air for 6 hours per day, 5 days/week for 90 days via nose-only inhalation. The lung burden and clearance with zirconium praseodymium yellow zircon 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 0.25, 0.15 and 0.08 mg/lung, in the mid dose groups 1.48, 1.28 and 1.16 mg/lung, and in the high dose groups 5.25, 4.76 and 3.87 mg/lung of the test item Zirconium Praseodymium Yellow Zircon (Pigment 5) were determined, respectively.

The clearance half-time of the test item amounted to 59 days in the low dose group, thus was 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 mid and high dose group, half-times of 277 and 210 days, respectively, were determined, being well above a 4- to 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. 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).

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:

Zirconium praseodymium yellow zircon

EC Number:

269-075-7

EC Name:

Zirconium praseodymium yellow zircon

Cas Number:

68187-15-5

Molecular formula:

Pr(x)Zr(1-x)SiO4 0,03≤x≤0,09

IUPAC Name:

silicon(4+) zirconium(4+) λ⁴-praseodymium(4+) dipraseodymium(3+) nonaoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Name: Colorante amarillo
- EC Name: Zirconium Praseodymium Yellow Zircon
- Substance type: inorganic pigment
- Physical state: solid, yellow powder, odourless
- 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.

*Reference:
- 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”) purchased from 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

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.18 - <= 2.21 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 three to 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 Zirconium praseodymium yellow zircon 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: ± 0.08 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: ± 0.38 mg/m³; 0.4 mg/lung (calculated total dose using MPPD v3.04)

Doses / concentrations 3

Dose / conc.:

9.99 mg/m³ air (analytical)

Remarks:

SD: ± 0.94 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.

FOOD EFFICIENCY: 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 (90 days post-exposure) - 10 (1 day post-exposure) per sex and dose group
- Parameters examined: total cell count, differential cell count (macrophages, neutrophils, eosinophils, lymphocytes; a total of 400 leukocytes per rat were evaluated), LDH, β-glucuronidase, and total protein

LUNG BURDEN: Yes
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
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 organ weights were caclulated.

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 5 (Zirconium praseodymium yellow zircon) high dose group after 90 days nose-only inhalation.
These organs were as follows: 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 will be performed in 10 animals per sex of the control and high dose group at day 1 post-exposure. Additionally, histopathological examination of animals of both sexes of the low and intermediate dose group is included.

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:

effects observed, treatment-related

Description (incidence and severity):

In males, statistically significant dose-dependent increases were observed for segmented (SEGC) and banded (BANC) neutrophils (calculated values) and proportions of lymphocytes and segmented neutrophils in the mid and high dose groups (please refer to: 'Attached background material'):
- The SEGC was increased by +36.5% and +59.8% in males of the intermediate and high dose group, respectively. The proportion of segmented neutrophils was increased by +31.4% and +35.4% in the intermediate and high dose group, respectively.
- The BANC was increased by +75.8% in males of the high dose group.
- The proportion of lymphocytes was decreased by -8.1% and -10.8% in males of the intermediate and high dose group respectively.

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):

Absolute and relative lung wet weights resulted dose-dependently in statistically significant increases in the mid and high dose groups of both sexes (please refer to: ‘Attached background material’):
- Males: One day after the last exposure, males exposed to 2.5 and 10 mg/m³ showed an increase of +11.7% and +18.8% in absolute lung wet weight, respectively. The relative weight was increased by +7.5% in the intermediate dose group and by +15.8% in the high dose group. At post-exposure day 92, the absolute weight was found to be increased by +22.4% and +20.7% in males of the intermediate and high dose group, respectively. The relative weight was found to be increased (+22.5%) only in males of the high dose group.
- Females: One day after the last exposure, females exposed to 2.5 and 10 mg/m³ showed an increase of +14.8% and +23.7% in absolute lung wet weight, respectively. The relative weight was increased by +15% in the intermediate dose group and by +22% in the high dose group. At post-exposure day 92, females of the high dose group showed absolute and relative wet lung weights increased by +21.4% and +18.7%, respectively.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Upon necropsy, enlarged lung-associated lymph nodes (LALN) were observed dose-dependently in the mid and high dose groups of both sexes (please refer to: ‘Attached background material’). This is a particle-specific lung clearance pathway. Lungs showed typical dose-dependent discolourations caused by the test item.
- One day after the last exposure, the LALN were found to be enlarged in 1/20, 5/20, 16/20, and 17/20 males exposed to 0, 0.6, 2.5, and 10 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the LALN were found to be enlarged in 2/10, 3/10, 9/10, and 10/10 animals, respectively. At post-exposure day 90, the LALN were enlarged in 0/5, 1/5, 4/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 1/5, 5/5, and 5/5 in the control, low, intermediate, and high dose group, respectively.
- One day after the last exposure, the lungs showed discoloured areas in 0/20, 6/20, 4/20, and 4/20 males exposed to 0, 0.6, 2.5, and 10 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the lungs showed discoloured areas in 1/10, 3/10, 2/10, and 3/10 animals, respectively. At post-exposure day 90, the lungs showed discolouration in 0/5, 2/5, 1/5, and 2/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 1/5, 0/5, and 1/5 in the control, low, intermediate, and high dose group, respectively.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings related to the inhalation of the test item were detected within the lung, the nasal cavity, and the lung-associated lymph nodes.

- Animals exposed to 0.6 mg/m³:
• In the lung, exposure-related findings represent very slight multifocal particle-laden macrophages in the alveoli in 10/10 males and 10/10 females.
• In the lung-associated lymph nodes (LALN), there was a very slight multifocal accumulation of particle-laden macrophages in 3/10 males and in 2/10 females.

- Animals exposed to 2.5 mg/m³:
• In the lung, exposure-related findings represent very slight multifocal granulocytic cell infiltration in the alveoli in 10/10 male and 9/10 female rats. In addition, in the lung alveoli, very slight multifocal extracellular material was seen in 2/10 female rats. This extracellular material appeared eosinophilic partially granular and was interpreted to be consisting of cell debris and surfactant admixed with particles. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (all slight) and 10/10 females (all slight) as well as in the bronchus-associated lymphoid tissue in 5/10 males (3 very slight; 2 slight) and in 6/10 females (5 very slight; 1 slight). This lesion correlated with a macroscopically observed dark grey multifocal discoloration of up to 1mm in diameter in the lung. Further in the lung interstitium, there was a multifocal very slight mixed inflammatory cell infiltration in 2/10 males and in 2/10 females. This interstitial inflammatory 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 females.
• 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 accumulation of particle-laden macrophages in 8/10 males (5 very slight; 3 slight) and in 9/10 females (3 very slight; 6 slight). In addition, 3 males (2 very slight; 1 slight) and 2 females (1 very slight; 1 slight) showed a diffuse increased cellularity of lymphocytes.
• These lesions correlated with a macroscopically observed enlargement and greenish discoloration in many animals.

- Animals exposed to 10 mg/m³:
• In the lung, exposure-related findings represent very slight multifocal granulocytic cell infiltration in the alveoli in 9/10 male and 10/10 female rats. In addition, in the lung alveoli, very slight multifocal extracellular material was seen in 7/10 male and 10/10 female rats. This extracellular material appeared eosinophilic partially granular and was interpreted to be consisting of cell debris and surfactant admixed with particles. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (all slight) and 10/10 females (1 very slight; 8 slight; 1 moderate) as well as in the bronchus-associated lymphoid tissue in 10/10 males (7 very slight; 3 slight) and in 9/10 females (6 very slight; 3 slight). This lesion correlated with a macroscopically observed dark grey multifocal discoloration of up to 1 mm in diameter in the lung. Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 3/10 males (all very slight) and in 5/10 females (4 very slight; 1 slight). This interstitial inflammatory 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 2/10 males and 2/10 females.
• In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 3/10 males and 4/10 females in the nose-associated lymphoid tissue (NALT).
• The occurrence of very slight multifocal hyaline droplets in 2 females was considered to be unrelated to the exposure.
• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 9/10 males (4 slight; 5 moderate) and in 10/10 females (4 slight; 6 moderate). In addition, 3 males (2 very slight; 1 slight) and 5 females (2 very slight; 3 slight) showed a diffuse increased cellularity of lymphocytes.
• These lesions correlated with a macroscopically observed enlargement and greenish discoloration in many animals.

Please refer to: ‘Attached background material’ for statistical intergroup comparison and detailed listing.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
- At days 1 and 90 post-exposure, statistically significant and dose-dependent increases of polymorphonuclear neutrophils (PMN) were detected in the mid and high dose groups. The PMN percentages (29% and 42%, males - 29% and 30%, females in the mid and high dose groups, respectively) demonstrate a moderate inflammatory potential as compared to clean air control data (3-4% PMN).
- At day 90 post-exposure, for lactic dehydrogenase, ß-glucuronidase and total protein statistically significant and dose-dependent increases were detected in the mid and high dose groups of both sexes; after 90 days of recovery, however, still statistically significant increases were observed in the mid and high dose group of females.
• Lactic dehydrogenase activity: At day 1 post-exposure, lactic dehydrogenase activity was 2.0- and 2.9-fold increased in the male intermediate and high dose group, respectively. In females, the lactic dehydrogenase activity was 2.6- and 3.2-fold increased, respectively. At post-exposure day 92, females of the intermediate and high dose group showed a 1.9- and 2.4-fold increased lactic dehydrogenase activity, respectively.
• ß-glucuronidase activity: At day 1 post-exposure, the ß-glucuronidase activity was 2.5- and 5.0-fold increased in males of the intermediate and high dose group, respectively. In females, the ß-glucuronidase activity was 2.9- and 4.2-fold increased over controls. At post-exposure day 92, females of the intermediate dose group showed a 2.4-fold increase above controls, while the high dose group showed a 3.9-fold increased ß-glucuronidase activity.
• Total protein concentration: At day 1 post-exposure, the total protein concentration was 1.7- and 2.3-fold increased in the male intermediate and high dose group, respectively. In females, the total protein concentration was 1.9- and 2.4-fold increased, respectively. At post-exposure day 92, females of the intermediate and high dose group showed a 1.7- and 2.0-fold increased total protein concentration as compared to the control group.

- Lung weights (BAL): In both sexes, statistically significant increases were observed in the mid (+7.5% and +15% in males and females, respectively, at post-exposure day 1) and high dose (+15.8% and +22% in males and females, respectively, at post-exposure day 1; +18.7% in females at post-exposure day 92) groups.

LUNG BURDEN ANALYSIS:
- The absolute lung weight was statistically significantly increased in males of the intermediate dose group at post-exposure day 92 (+22.4%) as well as in males of the high dose group at post-exposure day 1, 28 and 92 (+22.6%, +20.2%, and +20.7%, respectively). The relative lung weight was statistically significantly increased in males of the high dose group at post-exposure days 28 and 92 (+24.4% and +22.5%, respectively).
Please refer to IUCLID section 7.1.1 "k_Creutzenberg_2022_lung burden" for detailed information on results of lung burden analysis.

Details on results:

MORTALITY:
Rat #9122 (female of the intermediate dose group) died spontaneously (day 77) and was preserved for histopathological examination. It showed macroscopically a dark red 5 mm in diameter sized focus in the right hemisphere of the brain. This correlated histologically with a focal severe vascular hamartoma and a focal acute moderate hemorrhage into the brain parenchyma. These lesions are interpreted to be the cause of death of this animal and represent a developmental disorder without any relation to the exposure.

BODY WEIGHT AND WEIGHT CHANGES:
Relevant statistically significant changes were not observed in the treatment groups as compared to concurrent controls.

WATER CONSUMPTION:
- Some observed statistically altered values were considered as incidental findings as these effects were without a clear dose-dependency.

GROSS PATHOLOGY:
- Other test item- or dose-related macroscopical findings were not detected. Only some incidental macroscopic observations were obtained (please refer to: ‘Attached background material’).

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- Incidental increases were observed in the absolute (+26.8%) and relative (+23.1%) thymus weight of males exposed to 2.5 mg/m³ immediately after the last exposure. On post-exposure day 1, the left ovaries, of females exposed to 0.6 mg/m³, showed an absolute increase of +21% as well as an absolute and relative increase of +25.5% and +25.7%, respectively, in females exposed to 2.5 mg/m³. On post-exposure day 1, the relative uterus weight, of females exposed to 10 mg/m³, was decreased by -28.3%. Moreover, at post-exposure day 92, females exposed to 0.6 mg/m³ showed increased absolute weights of the left adrenal (+49.7%) and lung (+18.4%) as well as a decrease in the relative weight of the thymus (-21.3%). These alterations were only slight in magnitude, not consistent within/between animals, not dose-dependent, and without histopathological correlates. Thus, these effects are considered to be incidental and without toxicological relevance.

HAEMATOLOGICAL FINDINGS:
- In both sexes, no relevant statistically significant changes as compared to concurrent controls.
- The thromboplastin time was statistically significantly decreased by -8.2%, -7.0%, and -10.6% in males exposed to 0.6, 2.5, and 10 mg/m³, respectively. However, the change is only very slight and considered to be not toxicologically relevant.

CLINICAL BIOCHEMISTRY FINDINGS:
- In females, the chloride level was statistically significantly increased on post-exposure day 6 at all concentrations tested (+2.0%, +1.5%, and +1.9%, respectively) (please refer to: ‘Attached background material’). However, the effect was not concentration dependent and was not observed in males at any concentration tested. Moreover, the sodium level was statistically significantly increased (+0.9%) in females exposed to 10 mg/m³. The increase was, however, only marginal and not observed in males. Thus, these effects are considered to be incidental and without toxicological relevance.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- Clean air control group: 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.
- Animals exposed to 0.6 mg/m³: In the nasal cavity and trachea no lesions were seen.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

0.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Inflammatory response seen in the lung (BALF and histopathology) of mid and high dose group animals

2

Key result

Dose descriptor:

LOAEC

Effect level:

2.5 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: BALF

Remarks on result:

other: observed at lung overload conditions, in exceedance of the maximum tolerated dose (MTD)

Target system / organ toxicity

Critical effects observed:

not specified

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 zirconium praseodymium yellow zircon.
All animals (but one sudden death) 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, haematology, food and water consumption.
The lung weights were statistically significantly increased in the mid and high dose groups. The BALF analyses revealed persistent, statistically significant and dose-dependent increases of polymorphonuclear neutrophils (PMN) in the mid and high dose groups, demonstrating, based on the effect magnitude, a moderate inflammatory potential. Moreover, the lactic dehydrogenase activity, ß-glucuronidase activity and total protein concentration were persistently, statistically significantly and dose-dependently increased in BALF of the mid and high dose groups of both sexes. Adverse findings, as revealed via histopathological examination, representing very slight alveolar granulocytic cell infiltration, very slight alveolar extracellular material and very slight to slight interstitial mixed inflammatory cell infiltration, were detected within the investigated zirconium praseodymium yellow zircon high dose group and partially (very slight alveolar granulocytic cell infiltration) in the mid dose group. In the low dose group, no adverse findings were detected.

Under the conditions of this test, a NOAEC OF 0.6 mg/m³ was derived for both sexes based on the absence of any overt toxicity. The toxicity seen at the LOAEC of 2.5 mg/m³ is a typical response of an overload condition, no other toxicity was seen that may indicate a test item-specific toxicity. This is corroborated by a significant prolongation of the clearance half-time for the mid- and high-concentration animals of approx. 4- to 5-fold above the physiological 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.