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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 Fe plasma concentrations were observed, and only a minor fraction (<0.003%) of the total administered dose of Fe 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 a 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.125, 0.5 and 2 mg/m³ of high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix. Further examinations of these dose groups are still ongoing. The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct (see attached documentation in the study record for further information).

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:
Rats were selected because of their 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: 160.7 g - 167.7 g; females: 127.4 g - 140.0 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:
The route of administration was selected 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 nos.: 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 by ICP-OES were conducted to determine the concentration, stability and homogeneity of the test item in the formulations (Fraunhofer IME, report no. EBR-152/6-27/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).

The test item-vehicle mixture were initially diluted, filtrated, acidified and measured by ICP-OES. The following solutions were used to calibrate the instrument: blank, 1 μg/L, 2.5 μg/L, 5 μg/L, 7.5 μg/L, 10 μg/L, 20 μg/L, 25 µg/L, 30 mg/L, 40 µg/L, 50 μg/L, 75 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L (calibrations were adapted to matrix and to concentrations measured in pre measurement series). Calibrations were performed before each measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument (correlation coefficient has to be at least 0.995). Correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.999948. For each sample, at least three internal measurements were performed and the mean was calculated. Samples were diluted for adaption to the calibration matrix and to fit into the calibration curve.

Instrumental and analytical set-up for the ICP-OES instrument:
- Agilent 720 (Agilent Technologies, Waldbronn, Germany)
- Nebulizer: sea spray nebulizer from Agilent
- spray chamber: glass cyclonic spray chamber from Agilent
- carrier gas flow: 0.75 L/min
- RF power: 1200W
- Wavelengths:
Fe: 238.204 nm, 241.052 nm, 259.837 nm, and 259.940 nm

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645) (Chemische Analytik - Nachweis-, Erfassungs- und Bestimmungsgrenze unter Wiederholbedingungen – Begriffe, Verfahren, Auswertung; German version DIN 32645:2008-11. Beuth Verlag.):
LOD: 3 * standard deviation of calibration blank/slope of the calibration
LOQ: 3 * LOD
The resulting LODs/LOQs are as follows:
- LOD: 0.20 µg/L (Fe)
- LOQ: 0.60 µg/L (Fe)
- correlation coefficient: 0.999986 (Fe)
The certified reference materials as well as 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 certified value.
Selected samples were fortified with a known amount of iron (by standard addition of commercial standards) to determine the standard recovery of iron. For fortified test item-vehicle mixture samples, recoveries were 99.5% for Fe.

Results:
Dose verification:
nominal dose: 1,000 mg/kg bw pigment (94.4 mg/kg bw Fe)
Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Fe: 110 - 113
Anaylsis of homogenity (3samples):
Recovery [%]:
Fe: 102 - 108
Anaylsis of concentration (1sample):
Recovery [%]:
Fe: 93.3
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-152/6-27/y)
Urine and plasma samples were obtained at study termination. Urine and plasma samples were analysed for iron levels by ICP-OES.
- 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, 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 adverse changes in behaviour, external appearance or faeces were noted for the male and female rats treated with 1000 mg reaction mass of fumes, silica and diiron trioxide/kg bw/day or for the animals treated with the vehicle control.
- all male and female rats treated with 1000 mg reaction mass of fumes, silica and diiron trioxide/kg bw/day revealed reddish discoloured faeces as of test day 3 (not an adverse effect, since test item is a red powder).

MORTALITY
- none of the animals died prematurely during the study.

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 reaction mass of fumes, silica and diiron trioxide/kg bw/day (data within the normal range).

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 reaction mass of fumes, silica and diiron trioxide/kg bw/day or for the animals treated with the vehicle control.

HAEMATOLOGICAL FINDINGS
- no test item-related influence in haematological and coagulation parameters was noted for the male and female rats treated with 1000 mg reaction mass of fumes, silica and diiron trioxide/kg bw/day compared to the control group.

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 in biochemical parameters of test item-treated animals compared with the control animals were recorded (not test item-related findings):
males (test day 29): decreased bilirubin (control group: 2.48 ± 0.48 µmol/L vs. treatment group: 1.90 ± 0.16 µmol/L; p ≤ 0.05), and increased sodium (control group: 139.8 ± 0.4 µmol/L vs. treatment group: 140.8 ± 0.8 mmol/L; p ≤ 0.05)
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.

BEHAVIOUR (FUNCTIONAL FINDINGS)
- neurological screening performed did not reveal any test item-related influence in the male and female rats treated with 1000 mg test item/kg bw/day.
- examination results of the animals treated with the vehicle control were also in the normal range.
- statistically significant difference (p ≤ 0.05) in a neurological parameter of a test item-treated animal compared with the control animals was recorded (not test item-related finding):
males (test week 4): decreased body temperature (control group: 37.5 ± 0.2 °C vs. treatment group: 37.1 ± 0.2 °C; p ≤ 0.05)
However, the stated body temperature 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 test item/kg bw/day compared to the control group.
- statistically significant differences 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 relative brain weight (control group: 5.910 ± 0.173 g/kg bw vs. treatment group: 6.447 ± 0.420 g/kg bw; p ≤ 0.05), decreased absolute right epididymis weight (control group: 0.462 ± 0.022 g vs. treatment group: 0.406 ± 0.025 g; p ≤ 0.01), increased relative left testis weight (control group: 4.561 ± 0.266 g/kg bw vs. treatment group: 5.045 ± 0.348 g/kg bw; p ≤ 0.05), increased relative heart weight (control group: 3.795 ± 0.200 g/kg bw vs. treatment group: 4.094 ± 0.052 g/kg bw; p ≤ 0.05), decreased absolute spleen weight (control group: 0.710 ± 0.020 g vs. treatment group: 0.632 ± 0.068 g; p ≤ 0.05), decreased relative thymus weight (control group: 2.011 ± 0.103 g/kg bw vs. treatment group: 1.661 ± 0.090 g/kg bw;p ≤ 0.01), and decreased absolute thymus weight (control group: 0.674 ± 0.023 g vs. treatment group: 0.528 ± 0.048 g; p ≤ 0.01)
The absolute organ weights for the brain, kidneys and liver were statistical significantly increased in the treated animals in comparison with the control animals. However, the relative organ weight of the respective organ was unaffected and the reported values for the absolute 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

GROSS PATHOLOGICAL FINDINGS
- none of the male and female rats treated with 1000 mg reaction mass of fumes, silica and diiron trioxide/kg bw/day revealed any 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.

TOXICOKINETICS
Iron is of negligible bioavailability from the test substance Reaction mass of fumes, silica and diiron trioxidel: by recalculating the urine levels and setting them into relation to the administered dose of the element Fe, it is reasonable to assume that the majority of the dose (>99.9%) represents non-absorbable, “inert” pigment, likely to be excreted via faeces. Please also refer to the field "Attached background material" below.
Furthermore, there were either no appreciable or only negligible increases in blood plasma levels for the element iron.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
NOAEL (oral; rats) > 1000 mg reaction mass of fumes, silica and diiron trioxide/kg bw/day

No test item-related changes were observed for clinical signs, mortality, neurologically screening, body weight/body weight gain, food consumption, water consumption, haematology, clinical chemistry, organ weights, ophthalmology, gross pathology, and histopathology.
The uptake of iron during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.005% of the Fe dose was excreted via urine, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that Fe is not biologically available upon ingestion of the pigment Reaction mass of fumes, silica and diiron trioxide.
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:
start: 2020-07-30 and still ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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 only in guideline)
GLP compliance:
yes (incl. QA statement)
Remarks:
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.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

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

IN-LIFE DATES: From: 2020-08-04 To: 2021-02-17
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.15 - <= 1.82 µm
Remarks on MMAD:
MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: Parameters were recorded by 20-minute means The were set at 22°C + 2°C for temperature and 55% + 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to or exactly 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 0.125, 0.5 and 2 mg/m³ High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix 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.:
0.125 mg/m³ air (analytical)
Remarks:
SD: ± 0.01 mg/m³; 0.02 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
0.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.04 mg/m³; 0.07 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD: ± 0.08 mg/m³; 0.3 mg/lung (calculated total dose using MPPD v3.04)
No. of animals per sex per dose:
10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.125, 0.5 and 2 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 4.3% (rel. density=2.5, 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 0.125 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.125 mg/m³ x 4.3% = 0.03 mg/lung
Example for deposited mass at 0.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.5 mg/m³ x 4.3% = 0.10 mg/lung
Example for deposited mass at 2 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2 mg/m³ x 4.3% = 0.4 mg/lung
Retained massat 2 mg/m³: approx. 0.3 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 (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was determined for each group on a weekly basis.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Water consumption was determined for each group on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: No

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

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

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 90 days post-exposure
- Dose groups that were examined: all
- Number of animals: 5 (90 days post-exposure) - 10 (1 day post-exposure) per sex and dose group
- Parameters examined: total cell count, differential cell count (macrophages, neutrophils, eosinophils, lymphocytes; a total of 400 leukocytes per rat were evaluated), LDH, β-glucuronidase, and total protein

LUNG BURDEN: Yes (determination of lung:body weight ratio before and after treatment as well as chemical analysis)
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
Sacrifice and pathology:
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 4 (High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix) 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 7, respectively, were examined histopathologically: Nasal and paranasal cavities, pharynx (Laryngopharynx/nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology was performed in 10 animals per sex of the control and high dose group at day 1 post-exposure. Additionally, histopathological examination of animals of both sexes of the low and intermediate dose group is included and still ongoing.
Statistics:
Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.
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 and females, statistically significant dose-dependent increases were observed for leukocytes (WBC), segmented (SEGC) and banded (BANC) neutrophils in the mid and high dose groups (calculated and percentual values) (please refer to: 'Attached background material'):
- The WBC was statistically significantly increased in males and females of the intermediate (+35.5% and +42.2%, respectively) and high dose (+35.1% and +35.9%, respectively).
- The BANC was statistically significantly increased in males and females of the intermediate (+129.9% and +137.6%, respectively) and high dose (+121.4% and +152.7%, respectively).
- The SEGC was statistically significantly increased in males and females of the intermediate (+92.3% and +104.7%, respectively) and high dose (+121.1% and +125.6%, respectively).
Clinical biochemistry findings:
not specified
Description (incidence and severity):
Information will be added to the robust study summary upon availability
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
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’):
- One day after the last exposure, males exposed to 2 mg/m³ showed an increase of 92.5% in absolute lung wet weight. The relative lung weight was increased by 21% and 93.5% in the intermediate and high dose groups, when compared to the control group. At post-exposure day 92, the absolute lung weight was increased by 113.2%. The relative lung weight was increased by 125.3%.
- One day after the last exposure, females exposed to 0.5 and 2 mg/m³ showed statistically significant increases in the absolute lung wet weight (+24.0% and +86.6%). The relative lung weight was increased by 19.4% and 81% in the intermediate and high dose groups, when compared to the control group. At post-exposure day 92, the absolute lung weight was increased by 22.9% and 94.8% in the intermediate and high dose groups. The relative lung weight was increased by 20.3% and 89.3%.
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, 8/20, 20/20, and 20/20 males exposed to 0, 0.125, 0.5, and 2 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the LALN were found to be enlarged in 2/10, 5/10, 9/10, and 10/10 animals, respectively. At post-exposure day 90, the LALN were enlarged in 0/5, 3/5, 5/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 4/5, 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, 3/20, 5/20, and 20/20 males exposed to 0, 0.125, 0.5, and 2 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the lungs showed discoloured areas in 1/10, 1/10, 2/10, and 9/10 animals, respectively. At post-exposure day 90, the lungs showed discolouration in 0/5, 3/5, 0/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 0/5, 0/5, and 5/5 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 0.125 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 5/10 male (4 very slight; 1 slight) and 8/10 female rats (8 very slight). Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (10 very slight) and in 10/10 females (10 very slight) as well as in the bronchus-associated lymphoid tissue in 1/10 males (1 very slight) and in 1/10 females (1 very slight). In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 4/10 males (2 very slight; 1 slight; 1 moderate) and in 1/10 females (1 very slight). There was a multifocal very slight bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 1/10 males and a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 1/10 males.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 1/10 males (1 moderate) and in 1/10 females (1 very slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi.

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 2/10 males (2 very slight) and in 9/10 females (8 very slight; 1 slight). In addition, 1 male (1 slight) and 3 females (3 slight) showed a diffuse increased cellularity of lymphocytes.
•These lesions correlated with a macroscopically observed enlargement in all animals.

- Animals exposed to 0.5 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 10/10 male (4 very slight; 6 slight) and 10/10 female rats (6 very slight; 4 slight). In addition, in the lung alveoli, multifocal extracellular material was seen in 5/10 male (4 very slight; 1 slight) and 5/10 female rats (5 very slight). 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 (1 very slight; 9 slight) and in 10/10 females (3 very slight; 7 slight) as well as in the bronchus-associated lymphoid tissue in 8/10 males (5 very slight; 3 slight) and in 7/10 females (2 very slight; 5 slight). This lesion correlated with a macroscopically observed pale white multifocal discoloration of up to 1mm in diameter in the lung. In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 10/10 males (1 very slight; 8 slight; 1 moderate) and in 9/10 females (4 very slight; 4 slight; 1 moderate). There was a multifocal very slight bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 2/10 males and in 3/10 females as well as a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 1/10 males.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 7/10 males (6 very slight; 1 slight) and in 5/10 females (5 very slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi. In addition to the accumulation of particle-laden macrophages in the BALT, diffusely the lymphocytes were increased in numbers very slight in 1/10 males and 2/10 females.

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (1 very slight; 1 slight; 8 moderate) and in 10/10 females (3 slight; 5 moderate; 2 severe). This was accompanied by a central necrosis of the accumulation of particle-laden macrophages in 3/10 males (2 very slight; 1 slight) and 3/10 females (2 slight; 1 moderate). In addition, 8/10 males (4 slight; 4 moderate) and 9 females (3 very slight; 4 slight; 2 moderate) showed a diffuse increased cellularity of lymphocytes.
• These lesions correlated with a macroscopically observed enlargement in all animals.

- Animals exposed to 2 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 10/10 male (7 slight; 3 moderate) and 10/10 female rats (1 very slight; 6 slight; 3 moderate). In addition, in the lung alveoli, multifocal extracellular material was seen in 10/10 male (1 slight; 9 moderate) and 10/10 female rats (1 slight; 8 moderate; 1 severe). This extracellular material appeared eosinophilic partially granular and was interpreted to be consisting of cell debris and surfactant admixed with particles. The more severe cases represent a beginning lipoproteinosis. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (4 slight; 6 moderate) and in 10/10 females (7 slight; 3 moderate) as well as in the bronchus-associated lymphoid tissue in 10/10 males (1 very slight; 6 slight; 3 moderate) and in 10/10 females (5 very slight; 4 slight; 1 moderate). This lesion correlated with a macroscopically observed pale white multifocal discoloration of up to 1mm in diameter in the lung. In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 10/10 males (3 very slight; 5 slight; 2 moderate) and in 10/10 females (2 very slight; 8 slight). There was a multifocal bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 10/10 males (6 very slight; 4 slight) and in 10/10 females (5 very slight; 5 slight) and a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 5/10 males and in 3/10 females.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 10/10 males (1 very slight; 9 slight) and in 10/10 females (4 very slight; 6 slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi. In addition to the accumulation of particle-laden macrophages in the BALT, diffusely the lymphocytes were increased in numbers in 8/10 males (6 very slight; 2 slight) and 6/10 females (5 very slight; 1 slight).

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (3 moderate; 7 severe) and in 10/10 females (3 moderate; 7 severe). This was accompanied by a central necrosis of the accumulation of particle-laden macrophages in 10/10 males (6 very slight; 4 slight) and 9/10 females (3 very slight; 4 slight; 2 moderate). In addition, 9 males (1 very slight; 4 slight; 3 moderate; 1 severe) and 9 females (2 very slight; 3 slight; 4 moderate) showed a diffuse increased cellularity of lymphocytes.

• In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 1/10 females in the nose-associated lymphoid tissue (NALT).

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 all treatment groups (please refer to: ‘Attached background material’). The PMN percentages (in the range from 25% to 55%, males - 27% to 50%, females in the low, mid and high dose groups) demonstrate a strong inflammatory potential as compared to clean air control data (3-4% PMN) at the given low aerosol concentrations.
- At day 1 post-exposure, for lactic dehydrogenase, ß-glucuronidase and total protein statistically significant and dose-dependent increases were detected in the mid (but ß-glucuronidase) and high dose groups both sexes; after 90 days of recovery, however, still statistically significant increases in the high dose group:
- Lactic dehydrogenase activity: At day 1 post-exposure, lactic dehydrogenase activity was 3.7- and 7.6-fold increased in the male intermediate and high dose group, respectively. In females, the lactic dehydrogenase activity was 3.4- and 8.9-fold increased, respectively. At post-exposure day 92, females of the high dose group showed a 5.3-fold increased lactic dehydrogenase activity as compared to the control group.
- ß-glucuronidase activity: At day 1 post-exposure, the ß-glucuronidase activity was 31.5-fold increased in males of the high dose group, when compared to the negative control group value. In females, the ß-glucuronidase activity was 32.6-fold increased over controls. At post-exposure day 92, females of the high dose group showed a 15.1-fold increased ß-glucuronidase activity as compared to the control group.
- Total protein concentration: At day 1 post-exposure, the total protein concentration was 2.4- and 4.8-fold increased in the male intermediate and high dose group, respectively. In females, the total protein concentration was 2.2- and 5.7-fold increased, respectively. At post-exposure day 92, females of the high dose group showed a 4.4-fold increased total protein concentration as compared to the control group.

LUNG BURDEN ANALYSIS:
- The results of the chemical lung burden analysis will be submitted later.

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

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

HAEMATOLOGICAL FINDINGS:
- One day after the end of exposure, females exposed to 0.125 mg/m³ showed a marginal but statistically significantly increased haematocrit value (+4.3%). However, no such effect was observed at higher doses. Thus, the finding is considered to be of an incidental nature and without toxicological relevance. Moreover, the thromboplastin time showed a mild but statistically significant reduction in males exposed to 0.5 (-8.9%) and 2 mg/m³ (-7%). The decrease was, however, not dose dependent and is considered to be not toxicologically relevant.

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- Incidental increases were observed in the absolute weight of the left ovary of females exposed to 0.5 mg/m³ (post-exposure day 1; +26.2%), absolute and relative weight of the thymus of males exposed to 0.125 mg/m³ (post-exposure day 92; +39.7% and +32.6%, respectively), absolute and relative weight of the left adrenal of females exposed to 0. 5 mg/m³ (post-exposure day 92; +36.8% and +33.9%, respectively), absolute weight of the uterus of females exposed to 0.125 mg/m³ (post-exposure day 92; +14.3%), as well as increased relative weight of the left and right kidneys of females exposed to 0.5 mg/m³ (post-exposure day 92; +13% and +11%, respectively). 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.

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

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- Clean air control: In one animal of the control group, a lymphoma of the hematolymphoid system with infiltration of lymphoma cells into the bone marrow, liver, lung-associated, mandibular and mesenteric lymph nodes, and spleen was observed. This correlated with macroscopically observed enlarged spleen and lung-associated lymph nodes. In addition, single lesions were seen in different other organs, which represented commonly found background lesions in this rat strain.
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.
- Animals exposed to 0.125 and 0.5 mg/m³: In the nasal cavity and trachea no lesions were seen.
- Animals exposed to 2 mg/m³: The occurrence of very slight multifocal hyaline droplets in 2 males was considered to be unrelated to the exposure.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
- Lung weights: In both sexes, no statistically significant changes as compared to concurrent controls.
Remarks on result:
not determinable
Remarks:
The NOAEC will be derived upon completeness of the data set.
Critical effects observed:
no
Conclusions:
In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.125, 0.5 and 2 mg/m³ of high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix.
All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed.
Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption.
Haematology showed statistically significant dose-dependent increases for leukocytes, segmented and banded neutrophils in the mid and high dose groups. The lung wet weight was dose-dependently in 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 all treatment groups demonstrating, based on the effect magnitude, a strong inflammatory potential. Moreover, lactate dehydrogenase (LDH), ß-glucuronidase and total protein showed persistent (in females), statistically significant and dose-dependent increases in the mid (but ß-glucuronidase) and high dose groups.
Adverse findings, as revealed by histopathology, representing very slight to moderate alveolar granulocytic cell infiltration, very slight to severe alveolar extracellular material, very slight to moderate lung interstitial mixed inflammatory cell infiltration, and very slight to moderate necrosis of particle-laden macrophages in the lung-associated lymph nodes, were detected within the investigated high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix mid and high dose group and partially in the low dose group.

The NOAEC will be derived upon completeness of the data set.

The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct. Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:
(i) chemical analysis of lung burden
(ii) clinical chemistry
(iii) QAU evaluation and study finalisation
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:
start: 2020-07-30 and still ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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 only in guideline)
GLP compliance:
yes (incl. QA statement)
Remarks:
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.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

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

IN-LIFE DATES: From: 2020-08-04 To: 2021-02-17
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.15 - <= 1.82 µm
Remarks on MMAD:
MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: Parameters were recorded by 20-minute means The were set at 22°C + 2°C for temperature and 55% + 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to or exactly 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 0.125, 0.5 and 2 mg/m³ High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix 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.:
0.125 mg/m³ air (analytical)
Remarks:
SD: ± 0.01 mg/m³; 0.02 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
0.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.04 mg/m³; 0.07 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
2 mg/m³ air (analytical)
Remarks:
SD: ± 0.08 mg/m³; 0.3 mg/lung (calculated total dose using MPPD v3.04)
No. of animals per sex per dose:
10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.125, 0.5 and 2 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 4.3% (rel. density=2.5, 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 0.125 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.125 mg/m³ x 4.3% = 0.03 mg/lung
Example for deposited mass at 0.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.5 mg/m³ x 4.3% = 0.10 mg/lung
Example for deposited mass at 2 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2 mg/m³ x 4.3% = 0.4 mg/lung
Retained massat 2 mg/m³: approx. 0.3 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 (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was determined for each group on a weekly basis.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Water consumption was determined for each group on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: No

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

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

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 90 days post-exposure
- Dose groups that were examined: all
- Number of animals: 5 (90 days post-exposure) - 10 (1 day post-exposure) per sex and dose group
- Parameters examined: total cell count, differential cell count (macrophages, neutrophils, eosinophils, lymphocytes; a total of 400 leukocytes per rat were evaluated), LDH, β-glucuronidase, and total protein

LUNG BURDEN: Yes (determination of lung:body weight ratio before and after treatment as well as chemical analysis)
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
Sacrifice and pathology:
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 4 (High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix) 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 7, respectively, were examined histopathologically: Nasal and paranasal cavities, pharynx (Laryngopharynx/nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology was performed in 10 animals per sex of the control and high dose group at day 1 post-exposure. Additionally, histopathological examination of animals of both sexes of the low and intermediate dose group is included and still ongoing.
Statistics:
Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.
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 and females, statistically significant dose-dependent increases were observed for leukocytes (WBC), segmented (SEGC) and banded (BANC) neutrophils in the mid and high dose groups (calculated and percentual values) (please refer to: 'Attached background material'):
- The WBC was statistically significantly increased in males and females of the intermediate (+35.5% and +42.2%, respectively) and high dose (+35.1% and +35.9%, respectively).
- The BANC was statistically significantly increased in males and females of the intermediate (+129.9% and +137.6%, respectively) and high dose (+121.4% and +152.7%, respectively).
- The SEGC was statistically significantly increased in males and females of the intermediate (+92.3% and +104.7%, respectively) and high dose (+121.1% and +125.6%, respectively).
Clinical biochemistry findings:
not specified
Description (incidence and severity):
Information will be added to the robust study summary upon availability
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
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’):
- One day after the last exposure, males exposed to 2 mg/m³ showed an increase of 92.5% in absolute lung wet weight. The relative lung weight was increased by 21% and 93.5% in the intermediate and high dose groups, when compared to the control group. At post-exposure day 92, the absolute lung weight was increased by 113.2%. The relative lung weight was increased by 125.3%.
- One day after the last exposure, females exposed to 0.5 and 2 mg/m³ showed statistically significant increases in the absolute lung wet weight (+24.0% and +86.6%). The relative lung weight was increased by 19.4% and 81% in the intermediate and high dose groups, when compared to the control group. At post-exposure day 92, the absolute lung weight was increased by 22.9% and 94.8% in the intermediate and high dose groups. The relative lung weight was increased by 20.3% and 89.3%.
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, 8/20, 20/20, and 20/20 males exposed to 0, 0.125, 0.5, and 2 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the LALN were found to be enlarged in 2/10, 5/10, 9/10, and 10/10 animals, respectively. At post-exposure day 90, the LALN were enlarged in 0/5, 3/5, 5/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 4/5, 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, 3/20, 5/20, and 20/20 males exposed to 0, 0.125, 0.5, and 2 mg/m³, respectively. In females of the control, low, intermediate, and high dose group the lungs showed discoloured areas in 1/10, 1/10, 2/10, and 9/10 animals, respectively. At post-exposure day 90, the lungs showed discolouration in 0/5, 3/5, 0/5, and 5/5 males of the control, low, intermediate, and high dose group respectively. In females, the incidences were 0/5, 0/5, 0/5, and 5/5 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 0.125 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 5/10 male (4 very slight; 1 slight) and 8/10 female rats (8 very slight). Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (10 very slight) and in 10/10 females (10 very slight) as well as in the bronchus-associated lymphoid tissue in 1/10 males (1 very slight) and in 1/10 females (1 very slight). In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 4/10 males (2 very slight; 1 slight; 1 moderate) and in 1/10 females (1 very slight). There was a multifocal very slight bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 1/10 males and a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 1/10 males.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 1/10 males (1 moderate) and in 1/10 females (1 very slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi.

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 2/10 males (2 very slight) and in 9/10 females (8 very slight; 1 slight). In addition, 1 male (1 slight) and 3 females (3 slight) showed a diffuse increased cellularity of lymphocytes.
•These lesions correlated with a macroscopically observed enlargement in all animals.

- Animals exposed to 0.5 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 10/10 male (4 very slight; 6 slight) and 10/10 female rats (6 very slight; 4 slight). In addition, in the lung alveoli, multifocal extracellular material was seen in 5/10 male (4 very slight; 1 slight) and 5/10 female rats (5 very slight). 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 (1 very slight; 9 slight) and in 10/10 females (3 very slight; 7 slight) as well as in the bronchus-associated lymphoid tissue in 8/10 males (5 very slight; 3 slight) and in 7/10 females (2 very slight; 5 slight). This lesion correlated with a macroscopically observed pale white multifocal discoloration of up to 1mm in diameter in the lung. In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 10/10 males (1 very slight; 8 slight; 1 moderate) and in 9/10 females (4 very slight; 4 slight; 1 moderate). There was a multifocal very slight bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 2/10 males and in 3/10 females as well as a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 1/10 males.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 7/10 males (6 very slight; 1 slight) and in 5/10 females (5 very slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi. In addition to the accumulation of particle-laden macrophages in the BALT, diffusely the lymphocytes were increased in numbers very slight in 1/10 males and 2/10 females.

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (1 very slight; 1 slight; 8 moderate) and in 10/10 females (3 slight; 5 moderate; 2 severe). This was accompanied by a central necrosis of the accumulation of particle-laden macrophages in 3/10 males (2 very slight; 1 slight) and 3/10 females (2 slight; 1 moderate). In addition, 8/10 males (4 slight; 4 moderate) and 9 females (3 very slight; 4 slight; 2 moderate) showed a diffuse increased cellularity of lymphocytes.
• These lesions correlated with a macroscopically observed enlargement in all animals.

- Animals exposed to 2 mg/m³:
• In the lung, exposure-related findings represent multifocal granulocytic cell infiltration in the alveoli in 10/10 male (7 slight; 3 moderate) and 10/10 female rats (1 very slight; 6 slight; 3 moderate). In addition, in the lung alveoli, multifocal extracellular material was seen in 10/10 male (1 slight; 9 moderate) and 10/10 female rats (1 slight; 8 moderate; 1 severe). This extracellular material appeared eosinophilic partially granular and was interpreted to be consisting of cell debris and surfactant admixed with particles. The more severe cases represent a beginning lipoproteinosis. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (4 slight; 6 moderate) and in 10/10 females (7 slight; 3 moderate) as well as in the bronchus-associated lymphoid tissue in 10/10 males (1 very slight; 6 slight; 3 moderate) and in 10/10 females (5 very slight; 4 slight; 1 moderate). This lesion correlated with a macroscopically observed pale white multifocal discoloration of up to 1mm in diameter in the lung. In addition, the macrophages in the alveolar were additionally increased in numbers (alveolar histiocytosis) multifocal in 10/10 males (3 very slight; 5 slight; 2 moderate) and in 10/10 females (2 very slight; 8 slight). There was a multifocal bronchiolo-alveolar hyperplasia of the alveolar type (pneumocytes type 2 hyperplasia) in 10/10 males (6 very slight; 4 slight) and in 10/10 females (5 very slight; 5 slight) and a multifocal very slight bronchiolo-alveolar hyperplasia of the bronchiolar type (bronchiolization) in 5/10 males and in 3/10 females.
• Further in the lung interstitium, there was a multifocal mixed inflammatory cell infiltration in 10/10 males (1 very slight; 9 slight) and in 10/10 females (4 very slight; 6 slight). This interstitial inflammatory cell infiltration was mainly observed in the vicinity of the terminal bronchi. In addition to the accumulation of particle-laden macrophages in the BALT, diffusely the lymphocytes were increased in numbers in 8/10 males (6 very slight; 2 slight) and 6/10 females (5 very slight; 1 slight).

• In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (3 moderate; 7 severe) and in 10/10 females (3 moderate; 7 severe). This was accompanied by a central necrosis of the accumulation of particle-laden macrophages in 10/10 males (6 very slight; 4 slight) and 9/10 females (3 very slight; 4 slight; 2 moderate). In addition, 9 males (1 very slight; 4 slight; 3 moderate; 1 severe) and 9 females (2 very slight; 3 slight; 4 moderate) showed a diffuse increased cellularity of lymphocytes.

• In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 1/10 females in the nose-associated lymphoid tissue (NALT).

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 all treatment groups (please refer to: ‘Attached background material’). The PMN percentages (in the range from 25% to 55%, males - 27% to 50%, females in the low, mid and high dose groups) demonstrate a strong inflammatory potential as compared to clean air control data (3-4% PMN) at the given low aerosol concentrations.
- At day 1 post-exposure, for lactic dehydrogenase, ß-glucuronidase and total protein statistically significant and dose-dependent increases were detected in the mid (but ß-glucuronidase) and high dose groups both sexes; after 90 days of recovery, however, still statistically significant increases in the high dose group:
- Lactic dehydrogenase activity: At day 1 post-exposure, lactic dehydrogenase activity was 3.7- and 7.6-fold increased in the male intermediate and high dose group, respectively. In females, the lactic dehydrogenase activity was 3.4- and 8.9-fold increased, respectively. At post-exposure day 92, females of the high dose group showed a 5.3-fold increased lactic dehydrogenase activity as compared to the control group.
- ß-glucuronidase activity: At day 1 post-exposure, the ß-glucuronidase activity was 31.5-fold increased in males of the high dose group, when compared to the negative control group value. In females, the ß-glucuronidase activity was 32.6-fold increased over controls. At post-exposure day 92, females of the high dose group showed a 15.1-fold increased ß-glucuronidase activity as compared to the control group.
- Total protein concentration: At day 1 post-exposure, the total protein concentration was 2.4- and 4.8-fold increased in the male intermediate and high dose group, respectively. In females, the total protein concentration was 2.2- and 5.7-fold increased, respectively. At post-exposure day 92, females of the high dose group showed a 4.4-fold increased total protein concentration as compared to the control group.

LUNG BURDEN ANALYSIS:
- The results of the chemical lung burden analysis will be submitted later.

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

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

HAEMATOLOGICAL FINDINGS:
- One day after the end of exposure, females exposed to 0.125 mg/m³ showed a marginal but statistically significantly increased haematocrit value (+4.3%). However, no such effect was observed at higher doses. Thus, the finding is considered to be of an incidental nature and without toxicological relevance. Moreover, the thromboplastin time showed a mild but statistically significant reduction in males exposed to 0.5 (-8.9%) and 2 mg/m³ (-7%). The decrease was, however, not dose dependent and is considered to be not toxicologically relevant.

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- Incidental increases were observed in the absolute weight of the left ovary of females exposed to 0.5 mg/m³ (post-exposure day 1; +26.2%), absolute and relative weight of the thymus of males exposed to 0.125 mg/m³ (post-exposure day 92; +39.7% and +32.6%, respectively), absolute and relative weight of the left adrenal of females exposed to 0. 5 mg/m³ (post-exposure day 92; +36.8% and +33.9%, respectively), absolute weight of the uterus of females exposed to 0.125 mg/m³ (post-exposure day 92; +14.3%), as well as increased relative weight of the left and right kidneys of females exposed to 0.5 mg/m³ (post-exposure day 92; +13% and +11%, respectively). 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.

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

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- Clean air control: In one animal of the control group, a lymphoma of the hematolymphoid system with infiltration of lymphoma cells into the bone marrow, liver, lung-associated, mandibular and mesenteric lymph nodes, and spleen was observed. This correlated with macroscopically observed enlarged spleen and lung-associated lymph nodes. In addition, single lesions were seen in different other organs, which represented commonly found background lesions in this rat strain.
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.
- Animals exposed to 0.125 and 0.5 mg/m³: In the nasal cavity and trachea no lesions were seen.
- Animals exposed to 2 mg/m³: The occurrence of very slight multifocal hyaline droplets in 2 males was considered to be unrelated to the exposure.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
- Lung weights: In both sexes, no statistically significant changes as compared to concurrent controls.
Remarks on result:
not determinable
Remarks:
The NOAEC will be derived upon completeness of the data set.
Critical effects observed:
no
Conclusions:
In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.125, 0.5 and 2 mg/m³ of high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix.
All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed.
Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption.
Haematology showed statistically significant dose-dependent increases for leukocytes, segmented and banded neutrophils in the mid and high dose groups. The lung wet weight was dose-dependently in 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 all treatment groups demonstrating, based on the effect magnitude, a strong inflammatory potential. Moreover, lactate dehydrogenase (LDH), ß-glucuronidase and total protein showed persistent (in females), statistically significant and dose-dependent increases in the mid (but ß-glucuronidase) and high dose groups.
Adverse findings, as revealed by histopathology, representing very slight to moderate alveolar granulocytic cell infiltration, very slight to severe alveolar extracellular material, very slight to moderate lung interstitial mixed inflammatory cell infiltration, and very slight to moderate necrosis of particle-laden macrophages in the lung-associated lymph nodes, were detected within the investigated high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix mid and high dose group and partially in the low dose group.

The NOAEC will be derived upon completeness of the data set.

The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct. Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:
(i) chemical analysis of lung burden
(ii) clinical chemistry
(iii) QAU evaluation and study finalisation
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 repeated dose toxicity study (LPT, 2017) was conducted in rats as a limit test to assess the effect of the pigment on rats following repeated oral administration. The study was performed 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 iron content. The comparison of the total administered final pigment dose of 1000mg/kg bw with the total mean Fe 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 iron fraction of <<0.001% for males and <0.005% for females. The Fe concentrations of plasma samples, collected from control and dose group animals at the day of sacrifice, were below 0.26 µg and 0.24 µg Fe /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 Fe plasma concentrations were observed, and only a minor fraction (<0.003%) of the total administered dose of Fe 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 0.125, 0.5 and 2 mg/m³ of high-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix. All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected. No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption. Haematology showed statistically significant dose-dependent increases for leukocytes, segmented and banded neutrophils in the mid and high dose groups. The lung wet weight was dose-dependently in 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 all treatment groups demonstrating, based on the effect magnitude, a strong inflammatory potential. Moreover, lactate dehydrogenase (LDH), ß-glucuronidase and total protein showed persistent (in females), statistically significant and dose-dependent increases in the mid (but ß-glucuronidase) and high dose groups. Adverse findings, as revealed by histopathology, representing very slight to moderate alveolar granulocytic cell infiltration, slight to severe alveolar extracellular material, very slight to slight lung interstitial mixed inflammatory cell infiltration, and very slight to moderate necrosis of particle-laden macrophages in the lung-associated lymph nodes. A NOAEC could not be derived due to the lack of the histopathology results from the low and intermediate dose group. The histopathological examination of these dose groups is still ongoing. The NOAEC will be derived upon availability of these data.

The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct. Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:

(i) chemical analysis of lung burden

(ii) clinical chemistry

(iii) QAU evaluation and study finalisation

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.The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

No classification for repeated dose toxocity according to EC Regulation No. 1272/2008 is anticipated.