High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

oxo[(oxoferrio)oxy]iron; silanedione

EC Number:

701-304-2

Cas Number:

1353091-50-5

Molecular formula:

(SiO2)(Fe2O3)

IUPAC Name:

oxo[(oxoferrio)oxy]iron; silanedione

Test material form:

solid: particulate/powder

Details on test material:

- Name: Colorante Rojo
- OLD EC Name: Reaction mass of fumes, silica and diiron trioxide
- NEW EC Name: High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix
- Substance type: inorganic pigment
- Physical state: solid, red powder, odourless
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

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

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

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.

Effect levels

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

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.