Zirconium praseodymium yellow zircon

Endpoint summary

Description of key information

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Pr plasma concentrations were observed, and only a minor fraction (<<0.001%) of the total administered dose of Pr was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

 

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. An inflammatory response was observed especially in the mid- and high-dose group animals, which are a cause of the significantly impaired lung clearance ocurring at overload conditions. This is corroborated by significantly increase clearance half-times exceeding the physiological values for these dose groups. A test-item-specific toxicity was not observed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-23 to 2015-04-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008-10-03

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry and stored in a tightly closed container.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 36 days; females: 36 days
- Weight at first dosing: males: 154.8 g - 171.6 g; females: 128.0 g - 143.2 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany).
- Diet (ad libitum): commercial ssniff® R/M-H V1530 diet (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: no contaminants above the limitiations were noted for drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 15 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

According to the expected route of exposure.

Vehicle:

other: 0.8 % aqueous hydroxyl propyl methylcellulose gel

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentration. The administration formulation was continuously agitated by stirring throughout the entire administration procedure.
The administration formulation was freshly prepared every day.
Administration volume: 10 mL/kg bw/day
The amount of the test item was adjusted to each animal's current body weight daily.

VEHICLE
- Source: FAGRON GmbH & Co. KG, 22885 Barsbüttel, Germany
- Batch no.: 13D03-N03

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the test item that was mixed with the vehicle, tests were conducted to determine the concentration, stability and homogeneity of the test item in the formulations (Fraunhofer IME, report no. EBR-153/6-27/y).
For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at ≤-20 °C:

1) At study initiation:
- analysis of stability and concentration: immediately after preparation of the administration formulation as well as after 8 and 24 hours storage at room temperature (number of samples: 3).
- homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of the test item treated group (number of samples: 3).

2) at study termination:
- analysis of concentration: during treatment always before administration to the last animal of the test item treated group (number of samples: 1).

Before measurement of the dissolved zirconium and praseodymium in the test item-vehicle mixture the samples were retrieved from the cryogenic storage container and thawed at least overnight. Afterwards the samples were prepared for the measurements. For this the samples have to be diluted with 10% HNO3 due to the fact, that a quantity of undissolved test item was visible in the test sample vials. After dilution the samples were filtered to remove particles from the diluted samples. The dissolved samples were measured by ICP-MS.

The ICP-MS measurements were performed using an Agilent 7700 ICP-MS (Agilent Technologies, Waldbronn, Germany). For the determination of zirconium the isotopes 90Zr, 91Zr and 92Zr were measured; for praseodymium the isotope 141Pr was measured (monoisotopic element). Depending on the concentration range of the samples, the following standard solutions were used for the calibrations: blank, 0.1 μg/L, 0.25 μg/L, 0.5 μg/L, 0.75 μg/L, 1.0 μg/L, 2.5 μg/L, 5.0 μg/L, 7.5 μg/L, 10.0 μg/L, 12.5 μg/L, 15 μg/L, 17.5 μg/L, 20 μg/L, 22.5 μg/L and 25 μg/L. The calibration was performed before the measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-MS
instrument software. The respective measurement mode (noGas, Helium mode or HiHelium mode) for interference-free measurement with recoveries in the range of the quality control parameters for the validation samples (certified reference material, quality control standards, recalibration standards, fortifications, digested certified reference material) in the measurement series and a correlation coefficient with at least 0.995 were used for calculating concentrations. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Concentric nebulizer, from Agilent
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Make-up/Dilution Gas gas flow: 0.16 L/min
RF power: 1500 W
Isotopes: 90Zr, 91Zr, 92Zr, 141Pr and 103Rh (internal standard)

The applied LOD/LOQs were calculated as follows:
LOD: 3 * standard deviation of calibration blank divided by the slope of calibration line;
LOQ: 3 * LOD

The resulting LODs/LOQs are as follows:
- LOD: 0.00002 - 0.0001 µg/L (Pr); 0.002 - 0.009 µg/L (Zr)
- LOQ: 0.00007 - 0.0004 µg/L (Pr); 0.007 - 0.027 µg/L (Zr)
- correlation coefficient: 0.9987951 - 0.999923 (Pr); 0.9997754 - 0.999969 (Zr)

The quality control standards and recalibration standards were analyzed as quality assurance samples along with the test samples. To meet quality assurance requirements recovery needs to be in the range of ± 15 % of the respective value.

Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Pr: 5.13 - 6.13
Zr: 0.014 - 0.016

Analysis of homogeneity (3 samples):
Recovery [%]:
Pr: 2.36 - 6.01
Zr: 0.009 - 0.013

Analysis of concentration (1 sample)
Recovery [%]:
Pr: 8.18
Zr: 0.016

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in agreement with the Sponsor and based on available toxicity data a limit test was performed.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs): before and after dosing at each time of dosing as well as regularly throughout the working day from 7.30 a.m. to 4.30 p.m. and on Saturdays and Sundays from 8.00 a.m. to 12.00 noon with a final check performed at approx. 4.00 p.m.
- Time schedule (mortality): early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays with a final check at approx 4.00 p.m.
- Cage side observations checked: clinical signs & mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once a week thereafter (1, 2, 4, 8 and 24 hours after administration) as well as in test week 4 prior to any laboratory investigations.

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter (always on the same day of the week)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.
The relative food consumption (in g/kg bw/day) was determined
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of administration and at the end of test week 4
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute; neutrophilic granulocytes, eosinophilic granulocytes, basophilic granulocytes, lymphocytes, monocytes, and large unstained cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 4 approx. 1 to 2 hours after dosing and before any blood sampling
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory reactivity / grip strength / motor activity
1) Observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotype, toe pinch, tail pinch, wire maneuver, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, and auditory function
2) Functional tests: grip strength and locomotor activity

IMMUNOLOGY: No

TOXICOKINETC: Yes (please refer to Fraunhofer IME, report no. EBR-153/6-27/y)
Urine and plasma samples were obtained at study termination. Urine and plasma samples were analysed for zirconium and praseodymium levels by ICP-MS.
- urine sample: individual urine samples were collected from all animals before scheduled sacrifice following the last administration on test day 28. The animals were placed in metabolic cages during a 24-hour collection period, directly after the last oral administration. The urine weight/animal was determined upon removal of the sample. Pooled blank urine were obtained from spare animals.
- plasma sample: on the scheduled day of sacrifice, a terminal blood sample was collected from all animals under isoflurane anaesthesia in order to obtain LiHeparin plasma/animal. Afterwards, the animals were sacrificed and dissected.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

On test day 29 (approx. one day after the last administration), the animals were sacrifice and macroscopically inspected. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

The weights of the following organs of all animals were determined before fixation: adrenal gland (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), spleen, testicle (2), thymus, as well as prostate and seminal vesicles with coagulating glands as a whole.
Paired organs were weighed individually and identified as left or right.

The following organs or parts of organs of all animals were fixed in 7% buffered formalin (exceptions: eyes fixed in Davidson's solution and testes in Bouin's solution): adrenal gland (2), bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer´s patches; Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland (male and female), muscle (skeletal, leg), nerve (sciatic), ovary (2), pituitary, prostate and seminal vesicles with coagulating glands, spinal cord (3 sections), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (incl. regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts), and vagina

The above-listed organs of all animals were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.

Statistics:

The test item-treated group was compared with the vehicle control group:
The following statistical methods were used:

1) STUDENT's t-test: all numerical functional tests / body weight / food consumption / haematology and coagulation / clinical biochemistry / relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
p = 0.05/0.01 about t = 2.3060/3.3554 (for 8 degrees of freedom)

2) Exact test of R. A. FISHER: histology (p ≤ 0.05 and p ≤ 0.01)

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS
- no changes in behaviour or external appearance were noted for the male and female rats treated with 1000 mg zirconium praseodymium yellow zircon/kg bw/day or for the animals treated with the vehicle control.
- faeces of the control and test item-treated animals were formed normally.

MORTALITY
- none of the animals died prematurely

BODY WEIGHT AND WEIGHT CHANGES
- no test item-related influence was observed for the body weight, the body weight gain and body weight at autopsy in the male and female rats treated with 1000 mg test item./day (data within the normal range).
- statistically significant differences in body weight of test item-treated animals compared to the control animals were recorded (not test item-related findings):
males (test week 0, 1, 2, 3, and 4): increased body weight (p ≤0.01 or p ≤ 0.05)

FOOD CONSUMPTION AND COMPOUND INTAKE
- no test item-related changes in relative food consumption were noted for the male and female rats treated with 1000 mg test item/kg bw/day compared to the control group.

WATER CONSUMPTION AND COMPOUND INTAKE
- visual appraisal of the drinking water consumption did not reveal any test item-related influence.

OPHTHALMOLOGICAL FINDINGS
- ophthalmological examination revealed no changes of the eyes and the optic region in the male and female rats treated with 1000 mg zirconium praseodymium yellow zircon/kg bw/day or for the animals treated with the vehicle control.
- no pathological changes were noted on the adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus

HAEMATOLOGICAL FINDINGS
- no test item-related influence on haematological and coagulation parameters was noted for the male and female rats treated with 1000 mg zirconium praseodymium yellow zircon/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in haematological parameters of test item-treated animals compared to the control animals were recorded (not test item-related findings):
females (test day 29): increased platelets (control group: 934.0 ± 115.5 x10³/µL vs. treatment group: 1134.8 ± 81.5 x10³/µL)
males (test day 29): increased absolute monocytes (control group: 0.138 ± 0.033 x10³/µL vs. treatment group: 0.200 ± 0.034 x10³/µL) and increased absolute basophilic granulocytes (control group: 0.024 ± 0.005 x10³/µL vs. treatment group: 0.038 ± 0.008 x10³/µL)
However, the stated haematological findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). These findings should therefore not be regarded as adverse response but as normal biological variation.

CLINICAL BIOCHEMISTRY FINDINGS
- no test item-related influence in biochemical parameters was noted for the male and female rats treated with 1000 mg test item/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in biochemical parameters of test item-treated animals compared to the control animals were recorded (not test item-related findings):
females (test day 29): decreased albumin (control group: 29.08 ± 1.03 g/L vs. treatment group: 27.74 ± 0.73 g/L)
males (test day 29): increased glucose (control group: 5.014 ± 0.330 mmol/L vs. treatment group: 5.794 ± 0.463 mmol/L) and decreased chloride (control group: 102.6 ± 0.5 mmol/L vs. treatment group: 101.4 ± 0.9 mmol/L)
However, the stated clinical chemistry findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). These findings should therefore not be regarded as adverse response but as normal biological variation.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
- no test item-related changes in relative and absolute organ weights were noted for the male and female rats treated with 1000 mg zirconium praseodymium yellow zircon/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in organ weights of test item-treated animals compared with the control animals were recorded (not test item-related findings):
males (test day 29): increased absolute left epididymis weight (control group: 0.398 ± 0.036 g vs. treatment group: 0.468 ± 0.041 g), increased absolut right testis weight (control group: 1.512 ± 0.087 g vs. treatment group: 1.664 ± 0.087 g), increased absolute left kidney weight (control group: 1.296 ± 0.108 g vs. treatment group: 1.446 ± 0.088 g), increased absolute right kidney weight (control group: 1.304 ± 0.056 g vs. treatment group: 1.454 ± 0.101 g), increased relative spleen weight (control group: 1.775 ± 0.129 g/kg bw vs. treatment group: 2.041 ± 0.179 g/kg bw), and increased absolute spleen weight (control group: 0.530 ± 0.066 g vs. treatment group: 0.660 ± 0.060 g).
females (test day 29): decreased relative heart weight (control group: 4.540 ± 0.309 g/kg bw vs. treatment group: 3.998 ± 0.306 g/kg bw)
The absolute and/or relative organ weights for the epididymis, testis, kidney, spleen and heart were statistical significantly different in the treated animals in comparison with the control animals. However, the reported values for the organ weights are within the normal range for that rat strain and age of the animals (see attached historical control data of the lab in the field "Attached background material" below). These findings should therefore not be regarded as adverse response but as normal biological variation.

BEHAVIOUR (FUNCTIONAL FINDINGS)
- neurological screening did not reveal any test item-related influence in the male and female rats treated with 1000 mg zirconium praseodymium yellow zircon/kg bw/day.
- examination results of the animals treated with the vehicle control were also in the normal range, although the values for the spontaneous motility appeared to be below the normal range.
- statistically significant differences (p ≤ 0.05) in neurological parameters of test item-treated animals compared to the control animals were recorded (not test item-related findings):
males (test week 4): decreased hindlimp grip strength (control group: 293.7 ± 16.9 g vs. treatment group: 226.7 ± 22.0 g)
The hindlimb grip strength was statistically significantly decreased in the treated animals compared with the control animals. The mean grip strength of the hindlimb of both control (293.7 g) and treated (226.7 g) animals are both in the mean historical control range (214.0 - 419.3 g). Based on this it is concluded that this finding should therefore not be regarded as adverse response but as normal biological variation.
Please also refer for the historical control data to the field "Attached background material" below.

GROSS PATHOLOGICAL FINDINGS
- none of the male and female rats treated with 1000 mg test item/kg bw/day revealed any test item-related macroscopic changes at necropsy on test day 29.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- histomorphological examination did not reveal any morphological changes which are considered to be related to the administration of the test item (no difference between the groups).
- inflammatory lesions in different organs are considered to be coincidental findings or spontaneous organ changes and are thus not test item-related (no differences were noted between the groups).
- fatty infiltration in the hepatocytes and in the tubular epithelial cells of the kidney in male and female rats of the control and the test item-treated group were within the physiological limits.
- involution of the thymus in the rats of both groups corresponded in type, incidence and severity to the age of the animals.
- coincidental findings from different organs in a small number of control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.
- no morphological differences were noted for any of the organs examined between the controls and the test item treated dose group. Type, incidence and severity of the lesions were comparable to the control animals.

OTHER
TOXICOKINETICS
The analyis of zirconium and praseodymium concentrations of 24 h-urine sampled after the last dosing demonstrated the negligible bioavailability of the test substance, with < 0.05 % of the dose being exceted via urine for both metals. Similar, there were either no appreciable or only negligible increases in blood plasma levels for both metals. Please also refer to the field "Attached background material" below.

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

NOAEL (oral; rats) > 1000 mg zirconium praseodymium yellow zircon/kg bw/day

No test item-related changes were observed for clinical signs, mortality, neurologically screening, body weight, food consumption, water consumption, haematology, clinical chemistry, organ weights, ophthalmology, gross pathology, and histopathology. The analyis of zirconium and praseodymium concentrations of 24 h-urine sampled after the last dosing demonstrated the negligible bioavailability of the test substance, with < 0.05 % of the dose being exceted via urine for both metals. Similar, there were either no appreciable or only negligible increases in blood plasma levels for both metals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

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

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

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

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 (analytical)

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:

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

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.

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.

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

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)

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

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

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

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

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 (analytical)

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:

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

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.

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.

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

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)

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Animal data – oral route

 

A 28-day repeat dose toxicity limit test in rats was conducted (LPT, 2019) to assess the effect of the pigment on rats following repeated oral administration. The study was conducted according to OECD test guideline 407, and in compliance with GLP. Male and female rats were administered with the pigment by oral gavage for 28 days at a dose of 1000 mg/kg bw/day in 0.8% aqueous hydroxyl propyl methylcellulose gel. A concurrent control group was administered untreated vehicle. No clinical signs of toxicity were observed, and no animals died during the administration period. No changes in bodyweight gains, food consumption, haematology, clinical chemistry, organ weights or macropathology were observed which could be attributed to treatment with the test compound. The histomorphological examination of rat organs did not reveal any morphological lesions attributable to the administration of the test item. There were no morphological differences between the control rats and the test item-treated animals. No adverse effects were observed on the male and female reproductive organs. Furthermore, individual 24-hour urine samples were collected from all animals after the last dosing prior to the scheduled sacrifice, and in addition plasma samples were collected from each animal at the day of sacrifice. The plasma and urine samples were analysed for total praseodymium content. The comparison of the total administered final pigment dose of 1000mg/kg bw with the total mean Pr content recovered in 24-urine samples, as calculated from the mean 24h-urine collection volumes of 21 ml for males and of 16.3 ml for females, would correspond to a total bioavailable praseodymium fraction of <<0.001% for males and <0.001% for females. The Pr concentrations of plasma samples, collected from control and dose group animals at the day of sacrifice, were below 0.01 µg/L and 0.001 µg/L plasma.

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Pr plasma concentrations were observed, and only a minor fraction (<<0.001%) of the total administered dose of Pr was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

 

 

Animal data – inhalation route

 

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.

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).

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. 5-fold above the physiological value. 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.

*References:

- Driscoll, K.E. and Borm, P.J.A., 2020. Expert workshop on the hazards and risks of poorly soluble low toxicity particles. Inhal Toxicol 32(2):53-62.

Justification for classification or non-classification

No signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days and after 90 -days nose-only inhalation up to the maximum tolerated concentration.

No classification for Specific Target Organ Toxicity-Repeated Exposure (STOT-RE) according to EC Regulation No. 1272/2008 is required.