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Description of key information

A subacute inhalation toxicity study for Fe3O4, Fe2O3 and FeOOH and a subchronic inhalation study for Fe3O4 as representative source substances for the iron oxide category members are available. Additionally, a short-term (5-day) inhalation study with two different grades of nanomaterials was conducted. Rats were exposed to 10 and 30 mg/m³ of smaller nano-sized Fe2O3 and 30 mg/m³ of larger nano-sized Fe2O3.


Sub-chronic repeated dose toxicity studies with Fe3O4, Fe2O3 and FeOOH as representative source substances for the iron oxide category members are available. A NOAEL of greater than 1000 mg/kg bw/day is derived for all three substances, based on a complete absence of adverse effects.


For repeated dose toxicity via the dermal route, no reliable studies are available for the iron oxide category.


Details on the category justification are given in the read-across document attached in IUCLID section 13.2.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Justification for type of information:
see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Remarks:
not specified in the publication
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Route of administration:
oral: gavage
Vehicle:
water
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 per group
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The changes of serum biochemical parameters in the Fe2O3 nanoparticle groups were sporadic and were of a small magnitude, indicating that these differences were not considered dose-related adverse effects of the nanoparticle treatments.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Conclusions:
In the current repeated dose toxicity study, iron oxide red in water was administered via gavage to groups of 12 female Sprague-Dawley rats at dose levels of 250, 500, and 1000 mg/kg bw/day. The administration occurred daily for a 13-week period.

Fe2O3 nanoparticles had no significant effects on body weight, mean daily food and water consumption, when compared with control groups. There were no treatment-related changes in haematological, serum biochemical parameters or histopathological observations. Some changes in organ weights were observed: decreases in weight of pituitary gland and liver and increases in weight of adrenal gland and testis. According to the authors, ‘these changes were sporadic without dose-dependent trends, indicating that they were not considered toxicologically relevant’. In blood and all tissues tested, including liver, kidney, spleen, lung and brain, the concentration of Fe showed no dose-associated response in comparison to the control groups. Iron concentrations in the urine of Fe2O3 nanoparticle-treated rats showed no significant differences compared to those of control animals. Although not statistically significant, the concentrations of Fe in the faeces of treated animals were found to be higher than those of the control groups. The authors stated that the subchronic oral dosing with Fe2O3 nanoparticles showed no systemic toxicity to rats. The NOAEL was established to be greater than 1000 mg/kg bw/day, the highest dose tested in rats receiving Fe2O3 nanoparticles by gavage.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2018-10-12 to 2019-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018-06-25
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2017-05-08
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in a tightly-closed, original container in a cool and dry place
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at first dosing (main study, recovery and satellite animals): males: 57 days; females: 58 days

- Weight at first dosing:
main study and recovery animals: males: 317.3 g - 369.6 g; females: 193.9 g - 247.3 g
satellite animals: males: 312.1 g - 374.8 g; females: 203.0 g - 251.9 g

- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm 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): a certified commercial diet (ssniff®-R/M-H V1530, ssniff® Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water

- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55% ± 10% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on route of administration:
According to expected route of exposure and international guidelines.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): test item-diet mixtures were freshly prepared once a week.

- Mixing appropriate amounts with (ssniff®-R/M-H V1530): the appropriate amount of test item was weighed into a glass container. Some of the test item and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test item was distributed in the diet. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled with study number, group number, sex, and dose and stored at +10°C to +25°C.

To maintain a constant dose level in relation to the animals’ body weight, the concentration in the diet was adjusted based on the mean group food consumption per sex. The concentration was adjusted on a weekly basis using the food consumption values from the previous week.

The control animals received the standard diet only
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-diet mixtures (0, 100, 300 and 1000 mg/kg bw/day groups) by ICP-OES , samples of approximately 10 g were taken at the following times and stored at 20 °C ± 10 %:

1) Concentration and homogeneity: in test weeks 1, 4, 8 and 13 one sample from each of three areas (top, middle and bottom) of the bucket (only one sample from the control); due to gender dependent body weight gain, the test item concentration was calculated and mixed separately for males and females (number of samples (including controls): 10/sex/week; total: 20/week)

2) Concentration and stability (left-over diet): in test weeks 1, 4, 8 and 13 left-over diet food which has been available to the animals for 7 days; due to gender dependent body weight gain, the test item concentration was calculated and mixed separately for males and females (number of samples (including controls): 4/sex/week; total: 8/week)

Method:
The quantification of the concentration of the test item was performed by analysis of iron content with inductively coupled plasma with optical emission spectrometry (ICP-OES) after an oxidative digestion.

The formulation samples (dose test item 100 mg/kg bw, 300 mg/kg bw and 1000 mg/kg bw) contained the test item with concentrations between 1176 mg/kg and 20875 mg/kg (0 mg/kg added for the control samples) in diet samples. The samples were defrosted and homogenised using a overhead shaker. Afterwards a sample weight was taken, weighed exactly and transferred into a digestion vessel for digestion. Lastly, the digested samples were diluted prior to ICP-OES.

Results:
The validity of the applied method concerning linearity, precision and accuracy has been proven.

The generated results provide information about the concentration and the homogeneity of the test item in diet samples. The blank diet sample (diet sample without the test item) showed a substantial amount of iron (mean 262 ppm). The results for the formulation samples were corrected by the blank iron content. The summarised recovery results are as follows:

Recovery (%):
- 100 mg/kg bw/day: 60 - 130
- 300 mg/kg bw/day: 61 - 119
- 1000 mg/kg bw/day: 77 - 152

Regarding the iron content of the blank diet sample and its relatively wide variation which was subtracted from the measured iron content of the formulation samples, the iron recovery values for the formulation samples are in a reasonable range.

The recovery rates for iron content from the test item in formulation samples were in the range of 60 % to 152 %. The iron content results were corrected considering the mean blank value of the diet sample (control samples) and the blank value of the reagents.
Duration of treatment / exposure:
Main study and recovery groups: 90 days
Satellite groups: 91 days
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
actual test item intake (calculated; main study and recovery groups): males: 102.8 mg/kg bw/day; females : 104.2 mg/kg bw/day
actual test item intake (calculated; satellite groups): males: 101.7 mg/kg bw/day; females : 102.4 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
actual test item intake (calculated; main study and recovery groups): males: 309.3 mg/kg bw/day; females : 312.2 mg/kg bw/day
actual test item intake (calculated; satellite groups): males: 306.1 mg/kg bw/day; females : 316.3 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
actual test item intake (calculated; main study and recovery groups): males: 1031.2 mg/kg bw/day; females : 1038.6 mg/kg bw/day
actual test item intake (calculated; satellite groups): males: 1014.4 mg/kg bw/day; females : 1031.9 mg/kg bw/day
No. of animals per sex per dose:
Main study: 10 animals/sex/group
Recovery groups (control group and high dose group only): 5 animals/sex/group
Satellite groups: 10 animals/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected by the sponsor based on available data and the results of a preliminary, 14 day palatability study.

Groups of five male and five female Crl:CD(SD) rats were administered Ferroxide® Black 86 via diet at dose levels of 300 and 1000 mg/kg bw/day (equivalent to 295.8 and 988.5 mg/kg bw/day for males and 314.4 and 1005.9 mg/kg bw/day for females, respectively. The treatment period lasted 14 days. A control receiving standard diet only was run concurrently. Clinical signs, mortality, body weight, food consumption, drinking water consumption, gross pathology and test item intake were measured/recorded.

No deaths and no signs of toxicity were noted. No statistically significant influence was observed on the body weight, the body weight gain, and the food and drinking consumption. No test item-related findings were noted at macroscopic inspection at necropsy.

Darkly discoloured faeces were noted in all male and female animals from test day 6 until termination of the study on test day 15. This occurrence of darkly discoloured faeces is considered to be caused by the colour characteristics of the test item.

- Satellite groups: satellite animals were used for iron content determination and satellite groups were used for 0, 100, 300 and 1000 mg/kg bw/day dose levels.
- Post-exposure recovery period: 4 weeks
Positive control:
not applicable
Observations and examinations performed and frequency:
NOTE: recovery groups were only employed for the control group and the high dose group (1000 mg/kg bw/day)

CAGE SIDE OBSERVATIONS: Yes (main study, recovery groups and satellite groups)
- Time schedule:
1) Clinical signs: at least once daily (preferably at the same time each day); Animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.

2) Mortality/moribundity: early in the morning and again in the afternoon of each working day and on saturdays and sundays a similar procedure was followed except that the final check was carried out at approximately 3:30 p.m.

- Cage side observations checked: clinical signs and mortality/moribundity

DETAILED CLINICAL OBSERVATIONS: Yes (main study, recovery groups and satellite groups)
- Time schedule: once before the first exposure and once a week thereafter (always on the first day of the test week)(at the same time each day); in test week 13 these observations were performed prior to any laboratory investigations.

BODY WEIGHT: Yes (main study, recovery groups and satellite groups)
- Time schedule for examinations: at the time of group allocation and once a week thereafter always on the same day of the week throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (main study, recovery groups and satellite groups):
The quantity of food left by individual animals was recorded on a daily 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 upon completion of a treatment week. From these data the food consumption (in g/kg bw/week) was determined using the following formula:

relative food consumption (g/kg bw/day) = ((total food given (g) - total food left (g))/(number of animal days* x body weight (kg))

* The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

- Compound intake calculated: Yes (main study, recovery groups and satellite groups)
Individual test item intake was calculated on a weekly basis throughout the
experimental period based on concentration in the diet, individual food intake and body weight.

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 (main study, recovery groups and satellite groups)
- Time schedule for examinations: daily (visual appraisal)

OPHTHALMOSCOPIC EXAMINATION: Yes (main study and recovery groups)
- Time schedule for examinations: before start of dosing and at the end of the dosing and recovery period.
- Dose groups that were examined: all animals of the main study and recovery groups
- Parameters checked: pupillary reflex, adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, cornea, anterior chamber, lens, vitreous body, and fundus (retina, optic disc)

HAEMATOLOGY: Yes (main study and recovery groups)
- Time schedule for collection of blood: at the end of test week 13 (before necropsy on test day 91; main study) or at the end of the recovery period
(before necropsy on test day 120; recovery groups)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study and recovery groups
- Parameters checked: haemoglobin content, erythrocytes, leucocytes, reticulocytes, platelets, haematocrit value, differential blood count (relative; neutrophilic, eosinophilic granulocytes, basophilic granulocytes, lymphocytes, and monocytes as well as large unstained cells), differential blood count (absolute; neutrophilic, eosinophilic granulocytes, basophilic granulocytes, lymphocytes, and monocytes as well as large unstained cells), mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes (main study and recovery groups)
- Time schedule for collection of blood: at the end of test week 13 (before necropsy on test day 91; main study) or at the end of the recovery period
(before necropsy on test day 120; recovery groups)
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study and recovery groups
- Parameters checked: albumin, bilirubin (total), cholesterol (total), HDL cholesterol, LDL cholesterol, creatinine, glucose, protein (total), urea (in blood), calcium, potassium, sodium, alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase

PLASMA/SERUM HORMONES/LIPIDS: Yes (main study and recovery groups)
- Time of blood sample collection: at the end of test week 13 (before necropsy on test day 91; main study) or at the end of the recovery period (before necropsy on test day 120; recovery groups)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study and recovery groups
- Parameters checked: triiodthyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH)

The serum samples were stored at -20°C±10% until analysis using ELISA.
The T3, T4 and TSH ELISA (commercial kit ; Instrument: Tecan Sunrise) were conducted.
Control hormone levels were considered to differentiate between incidental and treatment-related changes. Control coefficients of variation were kept below 25 for T3 and T4 and below 35 for TSH, if possible. Stability of T3, T4 and TSH under selected storage conditions were tested as part of the hormonal assay validation.

URINALYSIS: Yes (main study and recovery groups)
- Time schedule for collection of urine: at the end of test week 13 (on test day 91; main study) or at the end of the recovery period (on test day 120; recovery groups)
- Metabolism cages used for collection of urine: Yes, urine was collected for 16 hours in an URIMAX funnel cage.
- Animals fasted: Yes, overnight
- Parameters checked: volume, colour, turbidity, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (approx. values), nitrite and microscopic examination of deposits (epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, casts) and crystalluria)

The collection of urine was terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemistry examinations.

NEUROBEHAVIOURAL EXAMINATION: Yes (main study and recovery groups)
- Time schedule for examinations: test weeks 13 (main study) and 17 (recovery groups) (before any blood sampling for laboratory examinations)
- Dose groups that were examined: all main study and recovery groups
- Battery of functions tested: sensory activity / 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, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation and auditory function

2) Grip strength: fore- and hindlimb strength

3) Locomotor activity: number of movements

IMMUNOLOGY: Not specified

AUDITORY ACUITY: Yes (main study and recovery groups)
The auditory acuity was checked with a simple noise test. This was done for all main study animals and recovery animals before start of dosing and at the end of the dosing and recovery period.

IRON LEVEL ANALYSIS (main group and satellite animals)
The iron level in plasma and tissues (liver and spleen) were determined in the current study. For analysis of the plasma, approximately 1 x 100 μL Li-Heparin plasma per animal was obtained, a sufficient volume of blood preferably from the
vena jugularis under isoflurane anaesthesia from main study and satellite animals as follows:
- at dissection: 10 animals/sex/group
Plasma samples for determination of iron levels were stored frozen at -20 °C ±10 % until analysis.

Before measurement of iron in rat plasma, samples were retrieved from cryogenic storage container and thawed at least overnight. Afterwards the samples were prepared for microwave digestion with HNO3 to digest the proteins. Then, iron in digested samples was measured by ICP-OES.

Before measurement of total iron in rat tissue (spleen and liver), the samples were retrieved from cryogenic storage container and were directly homogenized by manual milling (mortar). After homogenization the samples were lyophilized till weight stability. Afterwards samples were prepared for microwave digestion. Then, iron in digested samples was measured by ICP-OES.

The ICP-OES measurements were performed with an Agilent 5110 ICP-OES (Agilent Technologies, Waldbronn, Germany). Iron was detected at the wavelengths 238.204 nm, 234.350 nm and 259.940 nm. The following solutions were used to calibrate the instrument (Fe concentrations vary for the analysed media): blank, 1 μg/L, 2.5 μg/L, 4.0 μg/L, 5.0 μg/L, 6.0 μg/L, 7.5 μg/L, 8.0 μg/L, 10 μg/L, 20 μg/L, 25 μg/L, 40 μg/L, 50 μg/L, 60 μg/L, 75 μg/L, 80 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L. Calibrations were performed
before each measurement and in the respective acid matrix. The linearity of the calibration was adequate for the lower and higher concentration range. In some measurement series, individual concentrations were excluded from the calibration by the instrument software since the nominal concentration was ± 25% of the measured concentration, i.e. mainly low concentrations for which the difference between background of blank and concentration was not high enough. The calibration formula was calculated using the linear regression algorithm
of the ICP-OES instrument. The correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.99996. Specific wavelengths for the data evaluation were selected based on the best recovery of iron in quality control samples (certified waters, recovery/fortification samples, etc.). Furthermore, the wavelengths were checked for possible interferences and wavelengths with a possible interference were not taken into account for a possible evaluation.

Agilent 5110, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer, from Glass Expansion
Spray chamber: Iso Mist with Twister Helix from Glass Expansion
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 12.0 L/min
Additional gas flow: 1.00 L/min
Carrier gas flow: 0.7 L/min
RF power: 1200W
Stabilization time of sample: 20 sec
Repetition time (three internal measurements per sample): 30 sec
Wavelengths Fe measured: 234.350 nm, 238.204 nm, 239.563 nm, 259.940 nm and 261.187 nm

At least three internal measurements for each sample were performed and the mean was calculated and printed by the instrument software.
The applied LOD/LOQ calculations are (according to DIN 32645)*:
LOD: 3 x standard deviation of calibration blank/slope of the calibration
LOQ: 3 x LOD

The resulting LODs/LOQs are as follows:
- LOD: 0.193 - 0.937 µg/L
- LOQ: 0.578 - 2.81 µg/L
- correlation coefficient: 0.99996 - 1.00000

The certified reference materials TMDA-70.2, quality control standards, recalibration standards and fortified samples were analysed for quality assurance (QA) along with test samples during each measurement series. To meet quality assurance requirements, recovery needs to be in the range of ± 15 % of the respective certified value or the nominal/calculated values. Recovery of all quality assurance samples was compliant (i.e. recovery was in the range of ± 15 % of respective certified or nominal/calculated values).

Furthermore, the digested solid certified reference materials were used for validating not only the digestion but although the measurement itself. To meet quality assurance requirements, recovery needs to be in the range of ± 20 % of the respective certified value.

*Reference:
- Chemische Analytik - Nachweis-. Erfassungs- und Bestimmungsgrenze unter
Wiederholbedingungen - Begriffe. Verfahren. Auswertung; German version DIN
32645:2008-11. Beuth Verlag.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (main study and recovery groups)
On test day 91, the main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on test day 120. At necropsy, the oestrus cycle of all main study females was determined by taking vaginal smears. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated, weighed, dissected and inspected macroscopically.

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 under suitable illumination. 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 wet weights of the following organs of all main study and recovery animals were determined before fixation and relative weights were calculated: adrenal gland (2), brain, epididymis, heart, kidney (2), liver, ovary (2), pituitary, spleen, testicle (2), thymus, thyroid (1), uterus (including cervix), prostate and seminal vesicles with coagulating glands (as whole).

HISTOPATHOLOGY: Yes (main study and recovery groups)
The following organs or parts of organs with the exception of the eyes and testicles of all main study and recovery animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution and the testicles in modified Davidson's solution for optimum fixation. A portion of the colon was processed for the quantification of aberrant crypt foci.

Tissues collected for preservation / histopathology: gross lesions, adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve and Harderian gland (2), heart (left and right ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum, including Peyer´s patches), kidney and ureter (2), liver (2 lobes), lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland (male and female), muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary and oviducts (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, parotid, sublingual), skin (left flank), spinal cord (3 sections: cervical, mid-thoracic, lumbar), spleen, stomach, testis (2), thymus, thyroid (2) (including parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (including larynx), urinary bladder, uterus (including cervix) and vagina.

The organs of all main study and recovery animals were examined histologically after preparation of haematoxylin-eosin stained paraffin sections. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined as well.

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.

Detailed histopathologic examination with special attention to the mucosas was performed on the following organs of all main study and recovery animals after preparation of haematoxylin-eosin stained paraffin sections: oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum.

HISTOPATHOLOGY: Yes (satellite groups)
On test day 92, the satellite animals were dissected following a randomisation scheme. Satellite animals were dosed until one day before sacrifice.

Immediately after blood withdrawal for determination of iron levels, the animals were sacrificed by carbon dioxide (CO2 inhalation), exsanguinated, weighed, dissected and inspected macroscopically. The same macroscopical examinations as described for main study and recovery animals was conducted.

Furthermore, the wet weights of the following organs of all satellite animals were determined (where possible) before further processing: liver and spleen. The organs were stored frozen at -20°C ±10% until analysis.

QUANTIFICATION OF ABERRANT CRYPT FOCI (non-GLP; all main study and recovery animals of the control group and high dose group)

A portion of the colon from 2 cm distal of the caecum to 2 cm proximal of the anus was taken and processed for the quantification of aberration crypt foci (ACF).

The colon was opened longitudinally with scissors and gently rinsed with 0.9% NaCl solution to remove the colon contents. The colon was cut into pieces of suitable size to fit into histological cassettes; the pieces were placed on numbered pieces of filter paper of the same size. The most cranial piece was put onto filter paper number 1; all following pieces on filter papers with ascending numbers going towards the rectum.

Filter papers of an individual animal were placed in one cassette in numerical order with the paper number 1 (the most cranial portion) located at the bottom. The colon portions had to be as flat as possible to facilitate later processing and evaluation. If needed to ensure the colon portions were pressed flat and did not bulge or roll in the closed cassette, rolls of filter paper were added to the cassettes.

The tissue cassettes were closed and labelled with study number and animal number, but prior to transfer for examination the samples were coded to ensure blinded-analysis. The cassettes were immersed in 5% buffered formalin.

The following staining protocol was used for all colon tissue specimens (up to five parts) to visualize aberrant crypt foci (ACF):
1. 0.5% Methylene Blue Stain solution was prepared with distilled water.
2. Colon specimens were separately stained in 12-well plates for 60 minutes
3. Colon specimens were rinsed in a first container of distilled water.
4. Colon specimens were rinsed in a first container of distilled water.
5. All colon specimens were directly analysed using a dissecting microscope Leica Wild MZ8 at 50x magnification and a KL1550 electronic lamp

The figures provided in the publication of Shwter et al. (2014)* were used as examples for ACF.

*Reference
- A. N. Shwter et al.: Research Paper 'Chemoprevention of colonic aberrant crypt foci by Gynura procumbens in rats', Journal of Ethnopharmacology 151 (2014) 1194-1201.
Statistics:
The test item groups (100, 300 and 1000 mg/kg bw/day) were compared to the control group.

The following statistical methods were used:
1) Multiple t-test based on DUNNETT, C. W. New tables for multiple
comparisons with a control. Biometrics, 482-491 (September 1964): body weight / food consumption / haematology / coagulation / clinical chemistry / thyroid hormones / urinalysis / relative and absolute organ weights / iron levels of plasma, spleen and liver samples (p ≤ 0.05 and p ≤ 0.01)

2) Exact test of R. A. FISHER (if applicable): histopathology (p ≤ 0.05)

The following settings were used for the statistical evaluation of the parametrical values captured by the computerized system (Provantis® Integrated Preclinical Software, version 10.2.1):

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

The following statistical methods were used for the data not captured with the Provantis system:

1) STUDENT's t-test:
Numerical functional tests: body temperature / hind leg splay / grip strength / spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
p = 0.05 / 0.01 Δ t = 2.0484 / 2.7633 (for 28 degrees of freedom)
p = 0.05 / 0.01 ^ t = 2.0687 / 2.8073 (for 23 degrees of freedom)

These statistical procedures were used for all data.
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
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
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:
no effects observed
Details on results:
NOTE: recovery groups were only employed for the control group and the high dose group (1000 mg/kg bw/day)

CLINICAL SIGNS
1) Behaviour, external appearance and faeces
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: dark discoloured faeces were noted for all male and female rats treated with the test item for 90 days as of test days 29 (low dose), 6 (intermediate and high dose) and 7 (high dose). The faeces of all animals were of normal consistency throughout the experimental period. The discoloured faeces is considered to be caused by the colour characteristics of test item and is not considered to be an adverse effect.

- 1000 mg/kg bw/day: the faeces of the male and female rats previously treated with 1000 mg/kg bw/day for 90 days were still dark discoloured at the beginning of the recovery period up to and including test day 95. Afterwards, no changes in behaviour, external appearance or faeces were noted. The discoloured faeces is considered to be caused by the colour characteristics of test item and is not considered to be an adverse effect.

Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: dark discoloured faeces were noted for all male and female rats treated with the test item for 91 days as of test days 29 (low dose) and 6 (intermediate and high dose). The faeces of all animals were of normal consistency throughout the experimental period. The discoloured faeces is considered to be caused by the colour characteristics of test item and is not considered to be an adverse effect.

2) Detailed clinical observations
Main study and recovery groups
Treatment and recovery period:
- 0, 100, 300 and 1000 mg/kg bw/day: all parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination (test day -1). Also, all male and female control animals revealed normal values for each parameter set examined throughout the course of the study.

- 100 mg/kg bw/day: none of the animals treated with the test substance for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.

- 300 mg/kg bw/day: a black discolouration of fur was noted for 1/10 male and 3/10 female animals treated with the test substance for 90 days in test week 3 and a dark discoloured tail in up to 3/10 male and 2/10 female animals starting in test week 6.

- 1000 mg/kg bw/day: a black or dark discolouration of fur, tail and/or ears was noted for all male animals and up to 11/15 female animals treated with the test substance for 90 days starting in test week 3.

The discoloured areas of 2 affected animals were carefully rinsed with lukewarm soap water to determine if the discolouration was caused by soiling with the test item due to grooming or due to an inner accumulation of the test item. The discolouration could be removed, hence, the discolouration was considered to be caused by grooming and the colour characteristics of the test item which was administered via the diet.

Recovery period:
- 1000 mg/kg bw/day: a dark discolouration of ears was still noted in test week 14, a black or dark discolouration of fur up to test week 16 and a dark discoloured tail up to test week 17 for the animals previously treated with 1000 mg/kg bw/day for 90 days.

MORTALITY
1) Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: none of the male and female rats treated with the test substance for 90 days died or had to be sacrificed prematurely during the 90-day treatment period.

- 1000 mg/kg bw/day: none of the male and female rats previously treated with 1000 mg/kg bw/day of the test substance for 90 days died or had to be sacrificed prematurely during the 4-week recovery period.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: none of the male and female rats treated with the test substance for 91 days died or had to be sacrificed prematurely during the 91-day treatment period.

BODY WEIGHT AND WEIGHT CHANGES
1) Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals treated with the test item for 90 days compared to the control group during the 90-day treatment period.

- 1000 mg/kg bw/day: no test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals previously treated with 1000 mg/kg bw/day via the diet for 90 days during the 4 week recovery period.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals treated with the test item for 91 days compared to the control group during the 91-day treatment period.

Statistically significant differences in body weights compared to the control, which are not considered to be test item-related were recorded, as follows:

- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in body weight was observed for female rats on test day 22. The slight alteration in comparison to control animals is without any biological relevance.

-1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in body weight was observed for female rats on test day 90. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

FOOD CONSUMPTION
1) Main study and recovery groups
Treatment and recovery period
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative food consumption of the male and female animals treated with the test substance for 90 days compared to the control group during the 90 day treatment period.

- 1000 mg/kg bw/day: no test item-related influence was noted on the relative food consumption of the male and female animals previously treated with 1000 mg/kg bw/day for 90 days compared to the control group during the 4-week recovery period.

Statistically significant differences in relative food consumption compared to the control, which are not considered to be test item-related were recorded, as follows:

- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in body weight of male rats was observed in test weeks 10, 11 and 12. The slight alteration in comparison to control animals is without any biological relevance.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01 or p ≤ 0.05) increase in body weight of male rats was observed in test weeks 6, 9, 10, 11 and 12. The slight alteration in comparison to control animals is without any biological relevance.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative food consumption of the male and female animals treated with the test item via the diet for 91 days compared to the control group during the
91-day treatment period.

Statistically significant differences in relative food consumption compared to the control, which are not considered to be test item-related were recorded, as follows:

- 100 mg/kg bw/day: a statistically significant (p ≤ 0.01) increase in body weight of female rats was observed in test week 12. The slight alteration in comparison to control animals is without any biological relevance.

-300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in body weight of male rats was observed in test week 5. The slight alteration in comparison to control animals is without any biological relevance.

-1000 mg/kg bw/day: a statistically significant (p ≤ 0.01) decrease in body weight of male rats was observed in test week 7. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

TEST ITEM INTAKE
1) Main study and recovery groups
Treatment period:
The calculation of the test item intake via the diet revealed a considerable
exposure of the male and female animals to the test item.

The mean test item intake per week ranged from 93.6 to 109.5 mg/kg bw/day,
from 265.9 to 331.3 mg/kg bw/day and from 887.6 to 1113.6 mg/kg bw/day
for the male animals and from 86.0 to 112.2 mg/kg bw/day, from 259.9 to
343.7 mg/kg bw/day and from 871.6 to 1116.3 mg/kg bw/day for the female
animals, both genders treated with 100, 300 or 1000 mg/kg bw/day via the diet, respectively.

2) Satellite groups
Treatment period:
The calculation of the test item intake via the diet revealed a considerable
exposure of the male and female animals to the test item.

The mean test item intake per week ranged from 88.2 to 109.6 mg/kg bw/day,
from 267.1 to 336.3 mg/kg bw/day and from 876.8 to 1081.4 mg/kg bw/day
for the male animals and from 85.1 to 113.0 mg/kg bw/day, from 275.3 to
348.9 mg/kg bw/day and from 861.6 to 1139.1 mg/kg bw/day for the female
animals, both genders treated with 100, 300 or 1000 mg/kg bw/day via the diet, respectively.

Please also refer to the field "Attached background material".

WATER CONSUMPTION
Main study, recovery groups and satellite groups:
The visual appraisal of the drinking water consumption did not reveal any test
item-related influence in any of the dose groups.

OPHTHALMOLOGICAL FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: ophthalmological examination did not reveal any changes of the eyes and the optic region in the animals treated with the test substance for 90 days at the end of the treatment period.

- 1000 mg/kg bw/day: no changes of the eyes and the optic region were noted for the animals previously treated with the test substance for 90 days at the end of the recovery period.

HAEMATOLOGICAL FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on any of the haematological parameters of the male and female rats treated with the test substance for 90 days compared to the control group at the end
of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on any of the haematological parameters of the male and female rats previously treated with the test item via the diet for 90 days compared to the control group at the end of the recovery period on test day 120.

However, statistically significant differences in haematological parameters compared to the control, which are not considered to be test item-related were found as follows:

- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in basophilic granulocytes (abs.) was observed for female rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in large unstained cells (abs.) was observed for female rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance and dose-dependency was lacking.

Please also refer to the field "Attached background material".

CLINICAL BIOCHEMISTRY FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on any of the clinical chemistry parameters of the male and female rats treated with the test item for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on any of the clinical chemistry parameters of the male and female rats previously treated with the test item for 90 days compared to the control group at the end of the recovery period on test day 120.

However, statistically significant differences in clinical chemistry parameters compared to the control, which are not considered to be test item-related were found as follows:

- 1000 mg/kg bw/day: statistically significant (p ≤ 0.05) increases in protein (males), sodium (males) and alkaline phosphatase (females) were observed on test day 120. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

ENDOCRINE FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on any of the thyroid hormone levels of the male and female rats treated with the test substance for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on any of the thyroid hormone levels of the male and female rats previously treated with the test substance for 90 days compared to the control group at the end of the recovery period on test day 120.

However, statistically significant differences in thyroid hormone levels compared to the control, which are not considered to be test item-related were found as follows:

- 1000 mg/kg bw/day: statistically significant (p ≤ 0.05) increases in thyroid hormone T4 levels were observed for female and male rats on test days 91 and 120, respectively. The slight alteration observed for the females in comparison to control animals is without any biological relevance. Considering the findings of the male rats, the effect is due to the relative low or high values observed for the control group.

Please also refer to the field "Attached background material".

URINALSIS
Main study and recovery groups
Treatment and recovery period
- 100, 300 and 1000 mg/kg bw/day: no test item-related changes in the urinary status were noted for the male and female rats treated with the test substance for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on the urinary status of the male and female rats previously treated with the test substance for 90 days compared to the control group at the end of the recovery period on test day 120.

However, statistically significant differences in urine parameters compared to the control, which are not considered to be test item-related were found as follows:

- 1000 mg/kg bw/day: statistically significant (p ≤ 0.05) increases and decrease in pH were observed for female rats on test days 91 and 120, respectively. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

BEHAVIOUR (FUNCTIONAL FINDINGS)
Main study and recovery groups
Treatment period and recovery period:
- 100, 300 and 1000 mg/kg bw/day: the neurological screening performed at the end of the treatment period in test week 13 did not reveal any test item-related influence in the male and female rats treated with the test item for 90 days, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.

- 1000 mg/kg bw/day: no test item-related influence was noted on the parameters of the neurological screening of the male and female animals previously treated with 1000 mg/kg bw/day for 90 days at the end of the 4 week recovery period.

However, statistically significant differences in neurological parameters compared to the control, which are not considered to be test item-related were found as follows:

Main study:
- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in hindleg splay was observed for female rats in test week 13. Furthermore, a statistically significant (p ≤ 0.05) decrease in forelimb grip strength and a statistically significant (p ≤ 0.05) increase in hindlimb grip strength was observed for male rats in test week 13. The slight alteration in comparison to control animals is without any biological relevance

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01) decrease in spontaneous motility was observed for male rats in test week 13. Effect is due to the relative low or high value observed for the control group.

Recovery groups:
- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in hindlimb grip strength was observed for male rats in test week 17. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

ORGAN WEIGHTS
1) Main study and recovery animals
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative and absolute organ weights of the male and female animals treated with the test substance for 90 days compared to the control group at the end of the treatment period on test days 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on the relative and absolute organ weights of the male and female animals previously treated with the test substance for 90 days at recovery sacrifice on test day 120.

However, statistically significant differences in relative and absolute organ weights compared to the control, which are not considered to be test item-related were found as follows:

Relative organ weights:
- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in heart weight was observed for female rats in test week 91. The slight alteration in comparison to control animals is without any biological relevance and lacking dose-dependency.

- 300 and 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase thyroid/parathyroid weight was observed for male rats in test week 91. Effect is due to the relative low value observed for the control group.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in liver weight was observed for female rats in test week 120. The slight alteration in comparison to control animals is without any biological relevance.

Absolute organ weights
- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in heart weight was observed for female rats in test week 91. The slight alteration in comparison to control animals is without any biological relevance and lacking dose-dependency.

- 300 and 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01 or p ≤ 0.05) in increase thyroid/parathyroid weight was observed for male rats in test week 91. Effect is due to the relative low value observed for the control group.

2) Satellite animals
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative and absolute liver and spleen weights of the male and female animals treated with the test substance for 91 days compared to the control group at the end of the treatment period on test days 92.

However, statistically significant differences in relative and absolute organ weights compared to the control, which are not considered to be test item-related were found as follows:

Relative organ weights
-100 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in spleen weight was observed for male rats in test week 92. Dose-dependency was lacking.

Absolute organ weights
-100 mg/kg bw/day: a statistically significant (p ≤ 0.01) increase in spleen weight was observed for male rats in test week 92. Dose-dependency was lacking.

Please also refer to the field "Attached background material".

GROSS PATHOLOGICAL FINDINGS
1) Main study and recovery animals
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: macroscopic inspection at necropsy did not reveal any test item-related changes in the organs or tissues of the animals treated with the test substance for 90 days at terminal sacrifice on test day 91.

- 1000 mg/kg bw/day: no test item-related changes were noted in the organs or tissues of the animals previously treated with the test substance for 90 days at recovery sacrifice on test day 120.

A small number of macroscopic findings such as changes in the epididymides (reduced in size), kidneys (enlarged, yellowish discoloured), liver (white focus), lungs (emphysematous), pituitary (enlarged), testes (reduced in size) and/or uterus (dilated, filled with clear liquid) were noted only in few individual animals of the test item-treated groups and the control group and were therefore considered to be incidental findings.

2) Satellite animals
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: macroscopic inspection at necropsy did not reveal any changes in the organs or tissues of the animals treated with the test substance for 91 days at terminal sacrifice on test day 92.

HISTOPATHOLOGICAL FINDINGS-NON-NEOPLASTIC
Treatment and recovery period:
- 0, 100, 300 and 1000 mg/kg bw/day: daily exposure to a diet containing the test item for a period of 90 days did not result in any gross findings or in histopathological findings in a broad range of organs and tissues.

AUDITORY EXAMINATION
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: there was no indication of any impairment to auditory acuity.

IRON LEVEL ANALYSIS (main group and satellite animals)
Plasma samples:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the iron plasma levels of the male and female rats treated with the test substance for 91 days compared to the control group at the end of the treatment period.

- 100 mg/kg bw/day: apparently slightly increased iron plasma levels were noted for the male and female animals treated with the test substance compared to the control group (males: 83% without background correction, 262% with background correction, statistically significant at p ≤ 0.01; females: 40% without background correction, 102% with background correction, statistically significant at p ≤ 0.05). However, these are considered to be incidental findings as no test item-related effects were noted for the intermediate and high dose groups. In addition, as documented in a separate toxicokinetic study in rats, iron plasma levels in rats are subject to circadian variation, and the differences seen in this study are within the boundaries of these circadian variations.

Spleen samples:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the iron concentrations in spleen samples of the male and female rats treated with the test item for 91 days compared to the control group at the end of the treatment period. No statistically significant differences were noted compared to the control group.

Liver samples:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the iron concentrations in liver samples of the male rats treated with the test item for 91 days compared to the control group at the end of the treatment period. The iron concentrations in the liver samples of the male animals treated with 1000 mg /kg bw/day via the diet for 91 days were slightly increased by 35% to 38% compared to the control group (statistically significant at p ≤ 0.01). The female animals treated with the test item for 91 days revealed slightly increased iron concentrations in the liver samples by 43% to 57% for the low dose, by 42% to 59% for the intermediate dose, and by 48% to 63% for the high dose compared to the control group at the end of the treatment period. All values were statistically significant at p ≤ 0.01 compared to the control group. No dose-response relationship was noted.

Please also refer to the field "Attached background material".

QUANTIFICATION OF ABERRANT CRYPT FOCI (non-GLP; all main study and recovery animals of the control group and high dose group)

Visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species.
Key result
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
In the current repeated dose toxicity study, Ferroxide® Black 86 was administered via diet to groups of 10 male and 10 female Crl:CD (SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred daily for a 90- day period. A control group receiving plain diet was run concurrently. Furthermore, recovery groups (n = 10 animals/sex/group; control group and high dose level only; recovery period: 4 weeks) and a satellite group (n = 10 animals/sex/group: treatment period: 91 days) were also run concurrently.

During the observation of animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, haematological findings, clinical chemistry findings, endocrinological findings, urinalysis findings, behaviour (functional findings), organ weights, gross pathology and histopathological findings. Furthermore, iron level analysis of plasma and tissues (spleen and liver) revealed no test item-related influence on iron concentration in plasma and spleen samples. However, the iron concentrations in liver tissue samples were slightly increased by 35% to 63% for the female animals treated with 100 or 300 mg/kg bw/day of the test item via the diet and for the male and female animals treated with 1000 mg/kg bw/day via the diet for 91 days compared to the control group at the end of the treatment period. All values were statistically significant at p ≤ 0.01 compared to the control group. However, no dose-response relationship was noted. Lastly, visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species.

Based on the findings, the no observed adverse effect level (NOAEL) for general toxicity is considered to be greater than 1000 mg/kg bw/day for male and female rats.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2018-09-18 to 2019-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2018-06-25
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2017-05-08
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in a tightly-closed, original container in a dry place at ambient temperature
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at first dosing (main study, recovery and satellite animals): males: 62 days; females: 62 days

- Weight at first dosing:
main study and recovery animals: males: 364.9 g - 470.9 g; females: 195.0 g - 290.7 g
satellite animals: males: 345.5 g - 470.0 g; females: 197.9 g - 263.7 g

- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm 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): a certified commercial diet (ssniff®-R/M-H V1530, ssniff® Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water

- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55% ± 10% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Details on route of administration:
According to expected route of exposure and international guidelines.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): test item-diet mixtures were freshly prepared once a week.

- Mixing appropriate amounts with (ssniff®-R/M-H V1530): the appropriate amount of test item was weighed into a glass container. Some of the test item and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test item was distributed in the diet. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled with study number, group number, sex, and dose and stored at +10°C to +25°C.

To maintain a constant dose level in relation to the animals’ body weight, the concentration in the diet was adjusted based on the mean group food consumption per sex. The concentration was adjusted on a weekly basis using the food consumption values from the previous week.

The control animals received the standard diet only
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-diet mixtures (0, 100, 300 and 1000 mg/kg bw/day groups) by ICP-OES , samples of approximately 10 g were taken at the following times and stored at 20 °C ± 10 %:

1) Concentration and homogeneity: in test weeks 1, 4, 8 and 13 one sample from each of three areas (top, middle and bottom) of the bucket (only one sample from the control); due to gender dependent body weight gain, the test item concentration was calculated and mixed separately for males and females (number of samples (including controls): 10/sex/week; total: 20/week)

2) Concentration and stability (left-over diet): in test weeks 1, 4, 8 and 13 left-over diet food which has been available to the animals for 7 days; due to gender dependent body weight gain, the test item concentration was calculated and mixed separately for males and females (number of samples (including controls): 4/sex/week; total: 8/week)

Method:
The quantification of the concentration of the test item was performed by analysis of iron content with inductively coupled plasma with optical emission spectrometry (ICP-OES) after an oxidative digestion.

The formulation samples (dose test item 100 mg/kg bw, 300 mg/kg bw and 1000 mg/kg bw) contained the test item with concentrations between 1111 mg/kg and 22917 mg/kg (0 mg/kg added for the control samples) in diet samples. The samples were defrosted and homogenised using a overhead shaker. Afterwards a sample weight was taken, weighed exactly and transferred into a digestion vessel for digestion. Lastly, the digested samples were diluted prior to ICP-OES.

Results:
The validity of the applied method concerning linearity, precision and accuracy has been proven.

The obtained results provide information about the concentration and the homogeneity of the test item in diet samples. The control sample (diet without the test item) showed an amount of iron (mean 237 ppm). The results for the formulation samples were corrected for the blank iron content. The summarised recovery results are as follows:

Recovery (%):
- 100 mg/kg bw/day: 60 - 147
- 300 mg/kg bw/day: 67 - 161
- 1000 mg/kg bw/day: 81 - 153

Regarding the iron content of the blank diet sample and its relatively wide variation which was subtracted from the measured iron content of the formulation samples, the iron recovery values for the formulation samples are in a reasonable range.

The iron content results were corrected considering the reagent blanks. The
corrected iron content results of the control samples were between 172 ppm and
403 ppm and the mean value was 237 ppm. These variations indicate that the
ferrous test item was not fully homogeneously distributed, most likely due to
different particle sizes.

The recovery rates for iron content from the test item in formulation samples were in the range of 60 % to 161 %.


Duration of treatment / exposure:
Main study and recovery groups: 90 days
Satellite groups: 91 days
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual test item intake (calculated; main study and recovery groups): males: 103.9 mg/kg bw/day; females : 103.4 mg/kg bw/day
actual test item intake (calculated; satellite groups): males: 105.5 mg/kg bw/day; females : 108.9 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
actual test item intake (calculated based on concentration in diet, food intake and body weight; main study and recovery groups): males: 310.2 mg/kg bw/day; females : 311.1 mg/kg bw/day
actual test item intake (calculated; satellite groups): males: 306.7 mg/kg bw/day; females : 311.9 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
actual test item intake (calculated; main study and recovery groups): males: 1043.7 mg/kg bw/day; females: 1043.5 mg/kg bw/day
actual test item intake (calculated; satellite groups): males: 1098.8 mg/kg bw/day; females : 1040.4 mg/kg bw/day
No. of animals per sex per dose:
Main study: 10 animals/sex/group
Recovery groups (control group and high dose group only): 5 animals/sex/group
Satellite groups: 10 animals/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected by the sponsor based on available data and the results of a preliminary, 14 day palatability study.

Groups of five male and five female Crl:CD(SD) rats were administered Iron oxide Sicovit® yellow 10 E172 via diet at dose levels of 300 and 1000 mg/kg bw/day (equivalent to 286 and 987 mg/kg bw/day for males and 301 and 1004 mg/kg bw/day for females, respectively).The treatment period lasted 14 days. A control receiving standard diet only was run concurrently. Clinical signs, mortality, body weight, food consumption, drinking water consumption, gross pathology and test item intake were measured/recorded.

No deaths and no signs of toxicity were noted. No influence was observed on the
body weight, the body weight gain, and the food and drinking consumption. No
test item-related findings were noted at macroscopic inspection at necropsy.

Pale faeces were noted in 1 of 5 male rats exposed to the lower dose (nominal
300 mg/kg bw/day) of the test item on test day 14 as well as in 3 of 5 male and
4 of 5 female rats exposed to the high dose (nominal 1000 mg/kg bw/day) of the test item from test day 6 until study termination. This dose-related occurrence of discoloured faeces is considered to be caused by the colour characteristics of the test item.

- Satellite groups: satellite animals were used for iron content determination and satellite groups were used for 0, 100, 300 and 1000 mg/kg bw/day dose levels.
- Post-exposure recovery period: 4 weeks
Positive control:
not applicable
Observations and examinations performed and frequency:
NOTE: recovery groups were only employed for the control group and the high dose group (1000 mg/kg bw/day)

CAGE SIDE OBSERVATIONS: Yes (main study, recovery groups and satellite groups)
- Time schedule:
1) Clinical signs: at least once daily (preferably at the same time each day); Animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.

2) Mortality/moribundity: early in the morning and again in the afternoon of each working day and on saturdays and sundays a similar procedure was followed except that the final check was carried out at approximately 3:30 p.m.

- Cage side observations checked: clinical signs and mortality/moribundity

DETAILED CLINICAL OBSERVATIONS: Yes (main study, recovery groups and satellite groups)
- Time schedule: once before the first exposure and once a week thereafter (always on the first day of the test week)(at the same time each day); in test week 13 these observations were performed prior to any laboratory investigations.

BODY WEIGHT: Yes (main study, recovery groups and satellite groups)
- Time schedule for examinations: at the time of group allocation and once a week thereafter always on the same day of the week throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (main study, recovery groups and satellite groups):
The quantity of food left by individual animals was recorded on a daily 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 upon completion of a treatment week. From these data the food
consumption (in g/kg bw/week) was determined using the following formula:

relative food consumption (g/kg bw/day) = ((total food given (g) - total food left (g))/(number of animal days* x body weight (kg))

* The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

- Compound intake calculated: Yes (main study, recovery groups and satellite groups)
Individual test item intake was calculated on a weekly basis throughout the
experimental period based on concentration in the diet, individual food intake and body weight.

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: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE: Yes (main study, recovery groups and satellite groups)
- Time schedule for examinations: daily (visual appraisal)

OPHTHALMOSCOPIC EXAMINATION: Yes (main study and recovery groups)
- Time schedule for examinations: before start of dosing and at the end of the dosing and recovery period.
- Dose groups that were examined: all animals of the main study and recovery groups
- Parameters checked: pupillary reflex, adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, cornea, anterior chamber, lens, vitreous body, and fundus (retina, optic disc)

HAEMATOLOGY: Yes (main study and recovery groups)
- Time schedule for collection of blood: at the end of test week 13 (before necropsy on test day 91; main study) or at the end of the recovery period
(before necropsy on test day 119; recovery groups)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study and recovery groups
- Parameters checked: haemoglobin content, erythrocytes, leucocytes, reticulocytes, platelets, haematocrit value, differential blood count (relative; neutrophilic, eosinophilic granulocytes, basophilic granulocytes, lymphocytes, and monocytes as well as large unstained cells), differential blood count (absolute; neutrophilic, eosinophilic granulocytes, basophilic granulocytes, lymphocytes, and monocytes as well as large unstained cells), mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes (main study and recovery groups)
- Time schedule for collection of blood: at the end of test week 13 (before necropsy on test day 91; main study) or at the end of the recovery period
(before necropsy on test day 119; recovery groups)
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study and recovery groups
- Parameters checked: albumin, bilirubin (total), cholesterol (total), HDL cholesterol, LDL cholesterol, creatinine, glucose, protein (total), urea (in blood), calcium, potassium, sodium, alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase

PLASMA/SERUM HORMONES/LIPIDS: Yes (main study and recovery groups)
- Time of blood sample collection: at the end of test week 13 (before necropsy on test day 91; main study) or at the end of the recovery period (before necropsy on test day 119; recovery groups)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study and recovery groups
- Parameters checked: triiodthyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH)

The serum samples were stored at -20°C±10% until analysis using ELISA.
The T3, T4 and TSH ELISA (commercial kit ; Instrument: Tecan Sunrise) were conducted.
Control hormone levels were considered to differentiate between incidental and treatment-related changes. Control coefficients of variation were kept below 25 for T3 and T4 and below 35 for TSH, if possible. Stability of T3, T4 and TSH under selected storage conditions were tested as part of the hormonal assay validation.

URINALYSIS: Yes (main study and recovery groups)
- Time schedule for collection of urine: at the end of test week 13 (on test day 91; main study) or at the end of the recovery period (on test day 119; recovery groups)
- Metabolism cages used for collection of urine: Yes, urine was collected for 16 hours in an URIMAX funnel cage.
- Animals fasted: Yes, overnight
- Parameters checked: volume, colour, turbidity, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (approx. values), nitrite and microscopic examination of deposits (epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, casts) and crystalluria)

The collection of urine was terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemistry examinations.

NEUROBEHAVIOURAL EXAMINATION: Yes (main study and recovery groups)
- Time schedule for examinations: test weeks 13 (main study) and 17 (recovery groups) (before any blood sampling for laboratory examinations)
- Dose groups that were examined: all main study and recovery groups
- Battery of functions tested: sensory activity / 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, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation and auditory function

2) Grip strength: fore- and hindlimb strength

3) Locomotor activity: number of movements

IMMUNOLOGY: Not specified

AUDITORY ACUITY: Yes (main study and recovery groups)
The auditory acuity was checked with a simple noise test. This was done for all main study animals and recovery animals before start of dosing and at the end of the dosing and recovery period.

IRON LEVEL ANALYSIS (main group and satellite animals)
The iron level in plasma and tissues (liver and spleen) were determined in the current study. For analysis of the plasma, approximately 1 x 100 μL Li-Heparin plasma per animal was obtained, a sufficient volume of blood preferably from the
vena jugularis under isoflurane anaesthesia from main study and satellite animals as follows:
- at dissection: 10 animals/sex/group
Plasma samples for determination of iron levels were stored frozen at -20 °C ± 10 % until analysis.

Before measurement of iron in rat plasma, samples were retrieved from cryogenic storage container and thawed at least overnight. Afterwards the samples were prepared for microwave digestion with HNO3 to digest the proteins. Then, iron in digested samples was measured by ICP-OES.

Before measurement of total iron in rat tissue (spleen and liver), the samples were retrieved from cryogenic storage container and were directly homogenized by manual milling (mortar). After homogenization the samples were lyophilized till weight stability. Afterwards samples were prepared for microwave digestion. Then, iron in digested samples was measured by ICP-OES.

The ICP-OES measurements were performed with an Agilent 720 and 5110 ICP-OES (Agilent Technologies, Waldbronn, Germany). Iron was detected at the wavelengths 238.204 nm and 239.563 nm. The following solutions were used to calibrate the instrument (Fe concentrations vary for the analysed media): blank, 1 μg/L, 2.5 μg/L, 5.0 μg/L, 7.5 μg/L, 10 μg/L, 25 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 performed
before each measurement and in the respective acid matrix. The linearity of the calibration was adequate for the lower and higher concentration range. In some measurement series, individual concentrations were excluded from the calibration by the instrument software since the nominal concentration was ± 25% of the measured concentration, i.e. mainly low concentrations for which the difference between background of blank and concentration was not high enough. The calibration formula was calculated using the linear regression algorithm
of the ICP-OES instrument. The correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.999915. Specific wavelengths for the data evaluation were selected based on the best recovery of iron in quality control samples (certified waters, recovery/fortification samples, etc.). Furthermore, the wavelengths were checked for possible interferences and wavelengths with a possible interference were not taken into account for a possible evaluation.

Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer, from Glass Expansion
Spray chamber: Iso Mist with Twister Helix from Glass Expansion
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 15.0 L/min
Additional gas flow: 1.50 L/min
Carrier gas flow: 0.75 L/min
RF power: 1200W
Stabilization time of sample: 15 sec
Repetition time (three internal measurements per sample): 30 sec
Wavelengths Fe measured: 234.350 nm, 238.204 nm, 238.863 nm, 239.563 nm, 240.489 nm, 259.940 nm, 261.187 nm, 262.567 nm, 263.105 and 263.132 nm

Agilent 5110, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer, from Glass Expansion
Spray chamber: Iso Mist with Twister Helix from Glass Expansion
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 12.0 L/min
Additional gas flow: 1.00 L/min
Carrier gas flow: 0.7 L/min
RF power: 1200W
Stabilization time of sample: 20 sec
Repetition time (three internal measurements per sample): 30 sec
Wavelengths Fe measured: 234.350 nm, 238.204 nm, 239.563 nm, 259.940 nm and 260.709 nm

At least three internal measurements for each sample were performed and the mean was calculated and printed by the instrument software.
The applied LOD/LOQ calculations are (according to DIN 32645)*:
LOD: 3 x standard deviation of calibration blank/slope of the calibration
LOQ: 3 x LOD

The resulting LODs/LOQs are as follows:
- LOD: 0.137 - 0.770 µg/L
- LOQ: 0.412 - 2.31 µg/L
- correlation coefficient: 0.99999 - 0.999996

The certified reference materials TMDA-70.2, quality control standards, recalibration standards and fortified samples were analysed for quality assurance (QA) along with test samples during each measurement series. To meet quality assurance requirements, recovery needs to be in the range of ± 15 % of the respective certified value or the nominal/calculated values. Recovery of all quality assurance samples was compliant (i.e. recovery was in the range of ± 15 % of respective certified or nominal/calculated values).

Furthermore the digested solid certified reference materials were used for validating not only the digestion but although the measurement itself. To meet quality assurance requirements, recovery needs to be in the range of ± 20 % of the respective certified value.

*Reference:
- Chemische Analytik - Nachweis-. Erfassungs- und Bestimmungsgrenze unter
Wiederholbedingungen - Begriffe. Verfahren. Auswertung; German version DIN
32645:2008-11. Beuth Verlag.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (main study and recovery groups)
On test day 91, the main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on test day 119. At necropsy, the oestrus cycle of all main study and recovery females was determined by taking vaginal smears. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated, weighed, dissected and inspected macroscopically.

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 under suitable illumination. 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 wet weights of the following organs of all main study and recovery animals were determined before fixation and relative weights were calculated: adrenal gland (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), pituitary, spleen, testicle (2), thymus, thyroid (1), uterus (including cervix), prostate and seminal vesicles with coagulating glands (as whole).

HISTOPATHOLOGY: Yes (main study and recovery groups)
The following organs or parts of organs with the exception of the eyes and testicles of all main study and recovery animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution and the testicles in modified Davidson's solution for optimum fixation. A portion of the colon was processed for the quantification of aberrant crypt foci.

Tissues collected for preservation / histopathology: gross lesions, adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve and Harderian gland (2), heart (left and right ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum, including Peyer´s patches), kidney and ureter (2), liver (2 lobes), lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland (male and female), muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary and oviducts (2), pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, parotid, sublingual), skin (left flank), spinal cord (3 sections: cervical, mid-thoracic, lumbar), spleen, stomach, testis (2), thymus, thyroid (2) (including parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (including larynx), urinary bladder, uterus (including cervix) and vagina.

The organs of all main study and recovery animals were examined histologically after preparation of haematoxylin-eosin stained paraffin sections. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined as well.

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.

Detailed histopathologic examination with special attention to the mucosas was performed on the following organs of all main study and recovery animals after preparation of haematoxylin-eosin stained paraffin sections: oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum.

HISTOPATHOLOGY: Yes (satellite groups)
On test day 92, the satellite animals were dissected following a randomisation scheme. Satellite animals were dosed until one day before sacrifice.

Immediately after blood withdrawal for determination of iron levels, the animals were sacrificed by carbon dioxide (CO2 inhalation), exsanguinated, weighed, dissected and inspected macroscopically. The same macroscopical examinations as described for main study and recovery animals was conducted.

Furthermore, the wet weights of the following organs of all satellite animals were determined (where possible) before further processing: liver and spleen. The organs were stored frozen at -20°C ±10% until analysis.

QUANTIFICATION OF ABERRANT CRYPT FOCI (non-GLP; all main study and recovery animals of the control group and high dose group)

A portion of the colon from 2 cm distal of the caecum to 2 cm proximal of the anus was taken and processed for the quantification of aberration crypt foci (ACF).

The colon was opened longitudinally with scissors and gently rinsed with 0.9% NaCl solution to remove the colon contents. The colon was cut into pieces of suitable size to fit into histological cassettes; the pieces were placed on numbered pieces of filter paper of the same size. The most cranial piece was put onto filter paper number 1; all following pieces on filter papers with ascending numbers going towards the rectum.

Filter papers of an individual animal were placed in one cassette in numerical order with the paper number 1 (the most cranial portion) located at the bottom. The colon portions had to be as flat as possible to facilitate later processing and evaluation. If needed to ensure the colon portions were pressed flat and did not bulge or roll in the closed cassette, rolls of filter paper were added to the cassettes.

The tissue cassettes were closed and labelled with study number and animal number, but prior to transfer for examination the samples were coded to ensure blinded-analysis. The cassettes were immersed in 5% buffered formalin.

The following staining protocol was used for all colon tissue specimens (up to five parts) to visualize aberrant crypt foci (ACF):
1. 0.5% Methylene Blue Stain solution was prepared with distilled water fresh for every 48 tissue samples.
2. Colon specimens were separately stained in 6-12-well tissue culture plates filled with the solution for 60 minutes
3. Colon specimens were rinsed in a first container of distilled water.
4. Colon specimens were rinsed in a first container of distilled water.
5. All colon specimens were directly analysed using a dissecting microscope Leica Wild MZ8 at 50x magnification and a KL1550 electronic lamp

The figures provided in the publication of Shwter et al. (2014)* were used as examples for ACF.

*Reference
- A. N. Shwter et al.: Research Paper 'Chemoprevention of colonic aberrant crypt foci by Gynura procumbens in rats', Journal of Ethnopharmacology 151 (2014) 1194-1201.
Statistics:
The test item groups (100, 300 and 1000 mg/kg bw/day) were compared to the control group.

The following statistical methods were used:
1) Multiple t-test based on DUNNETT, C. W. New tables for multiple
comparisons with a control. Biometrics, 482-491 (September 1964): body weight / food consumption / haematology / coagulation / clinical chemistry / thyroid hormones / urinalysis / relative and absolute organ weights / iron levels of plasma, spleen and liver samples (p ≤ 0.05 and p ≤ 0.01)

2) Exact test of R. A. FISHER (if applicable): histopathology (p ≤ 0.05)

The following settings were used for the statistical evaluation of the parametrical values captured by the computerized system (Provantis® Integrated Preclinical Software, version 10.2.1):

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

The following statistical methods were used for the data not captured with the Provantis system:

1) STUDENT's t-test:
Numerical functional tests: body temperature / hind leg splay / grip strength / spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
p = 0.05 / 0.01 Δ t = 2.0484 / 2.7633 (for 28 degrees of freedom)
p = 0.05 / 0.01 ^ t = 2.0687 / 2.8073 (for 23 degrees of freedom)

These statistical procedures were used for all data.
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
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
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:
no effects observed
Details on results:
NOTE: recovery groups were only employed for the control group and the high dose group (1000 mg/kg bw/day)

CLINICAL SIGNS
1) Behaviour, external appearance and faeces
Main study and recovery groups
Treatment and recovery period:
- 100 mg/kg bw/day: none of the male and female rats treated with the test item for 90 days revealed any changes of behaviour or external appearance during the 90-day treatment period.

- 300 mg/kg bw/day: yellow discoloured faeces were noted for all male and female rats treated with the test item for 90 days as of test day 39.

- 1000 mg/kg bw/day: yellow discoloured faeces were noted for 2/15 male animals treated with the test item for 90 days on test day 7 and for all male and female high dose rats as of test day 39.

The faeces of all animals were of normal consistency throughout the experimental period.

- 1000 mg/kg bw/day: faeces of the male and female rats previously treated with the test item for 90 days were still yellow discoloured at the beginning of the recovery period up to and including test day 94. Afterwards, no changes in behaviour, external appearance or faeces were noted.

Satellite groups
Treatment period:
- 100 mg/kg bw/day: none of the male and female rats treated with the test item for 91 days revealed any changes of behaviour or external appearance during the 91-day treatment period.

- 300 mg/kg bw/day: yellow discoloured faeces were noted for all male and female rats treated with the test item for 91 days as of test day 39.

- 1000 mg/kg bw/day: yellow discoloured faeces were noted for all male and female animals treated with the test item for 91 days as of test day 39.

The faeces of all animals were of normal consistency throughout the experimental period.

The dose-related occurrence of discoloured faeces is considered to be caused by the colour characteristics of the test item and is not considered an adverse effect.

2) Detailed clinical observations
Main study and recovery groups
Treatment and recovery period:
- 0, 100, 300 and 1000 mg/kg bw/day: all parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination (test day -1). Also, all male and female control animals revealed normal values for each parameter set examined throughout the course of the study.

- 100 and 300 mg/kg bw/day: none of the animals treated with the test item for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.

- 1000 mg/kg bw/day: a yellow discolouration of fur, faeces and/or tail was noted for several animals treated with the test item via the diet for 90 days starting in test week 3.

- 1000 mg/kg bw/day: no changes in external appearance, body posture, movement and coordination capabilities, and behaviour were noted for the animals previously treated with the test item for 90 days during the 4-week recovery period.

This dose-related occurrence of discoloured faeces, fur and/or tail is considered to be caused by the colour characteristics of the test item and is not considered an adverse effect.

MORTALITY
1) Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: none of the male and female rats treated with the test substance for 90 days died or had to be sacrificed prematurely during the 90-day treatment period.

- 1000 mg/kg bw/day: none of the male and female rats previously treated with 1000 mg/kg bw/day of the test substance for 90 days died or had to be sacrificed prematurely during the 4-week recovery period.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: none of the male and female rats treated with the test substance for 91 days died or had to be sacrificed prematurely during the 91-day treatment period.

BODY WEIGHT AND WEIGHT CHANGES
1) Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals treated with the test item for 90 days compared to the control group during the 90-day treatment period.

- 1000 mg/kg bw/day: no test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals previously treated with the test item for 90 days during the 4-week recovery period.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals treated with the test item for 91 days compared to the control group during the 91-day treatment period.

However, statistically significant differences in body weights of satellite animals compared to the control, which are not considered to be test item-related were found as follows:

- 100 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in body weight was observed for male rats on test day -12. Effect was observed before dosing.

Please also refer to the field "Attached background material".

FOOD CONSUMPTION
1) Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative food consumption of the male and female animals treated with the test substance for 90 days compared to the control group during the 90 day treatment period.

- 1000 mg/kg bw/day: no test item-related influence was noted on the relative food consumption of the male and female animals previously treated with 1000 mg/kg bw/day for 90 days compared to the control group during the 4-week recovery period.

However, statistically significant differences in relative food consumption compared to the control, which are not considered to be test item-related were found as follows:

- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in food consumption of male rats was observed in test week 11. The slight alteration in comparison to control animals is without any biological relevance.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in food consumption of male rats was observed in test weeks 9 and 11. Furthermore, a statistically significant (p ≤ 0.05) decrease in food consumption of female rats was observed in test weeks 14, 15 and 16. The slight alteration in comparison to control animals is without any biological relevance.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative food consumption of the male and female animals treated with the test item for 91 days compared to the control group during the 91-day treatment period.

However, statistically significant differences in relative food consumption compared to the control, which are not considered to be test item-related were found as follows:

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01 or p ≤ 0.05) increase in food consumption of male rats was observed in test weeks 1, 2, 4, 5 , 6, 7 and 12. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

TEST ITEM INTAKE
1) Main study and recovery groups
Treatment period:
The calculation of the test item intake via the diet revealed a considerable
exposure of the male and female animals to the test item.

The mean test item intake per week ranged from 76.8 to 116.8 mg/kg bw/day,
from 241.7 to 347.1 mg/kg bw/day and from 790.1 to 1134.8 mg/kg bw/day
for the male animals and from 83.9 to 113.8 mg/kg bw/day, from 259.6 to
350.2 mg/kg bw/day and from 869.9 to 1156.4 mg/kg bw/day for the female
animals, both genders treated with 100, 300 or 1000 mg/kg bw/day via the diet, respectively.

2) Satellite groups
Treatment period:
The calculation of the test item intake via the diet revealed a considerable
exposure of the male and female animals to the test item.

The mean test item intake per week ranged from 80.1 to 120.7 mg/kg bw/day,
from 235.8 to 338.6 mg/kg bw/day and from 833.8 to 1223.9 mg/kg bw/day
for the male animals and from 90.4 to 125.5 mg/kg bw/day, from 255.0 to
343.8 mg/kg bw/day and from 884.2 to 1181.5 mg/kg bw/day for the female
animals, both genders treated with 100, 300 or 1000 mg/kg bw/day via the diet, respectively.

Please also refer to the field "Attached background material".

WATER CONSUMPTION
1) Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: the visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.

2) Satellite groups
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: the visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.

OPHTHALMOLOGICAL FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: ophthalmological examination did not reveal any changes of the eyes and the optic region in the animals treated with the test item for 90 days at the end of the treatment period.

- 1000 mg/kg bw/day: no changes of the eyes and the optic region were noted for the animals previously treated with the test item for 90 days at the end of the recovery period.

HAEMATOLOGICAL FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on any of the haematological parameters of the male and female rats treated with the test item for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on any of the haematological parameters of the male and female rats previously treated with the test item for 90 days compared to the control group at the end of the recovery period on test day 119.

However, statistically significant differences in haematological parameters compared to the control, which are not considered to be test item-related were found as follows:

- 100 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in absolute eosinophilic granulocytes was observed for female rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance and lacking dose-dependency.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in absolute eosinophilic granulocytes was observed for female rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in activated partial thrombplastin time was observed for male rats on test day 119. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

CLINICAL BIOCHEMISTRY FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on any of the clinical chemistry parameters of the male and female rats treated with the test item for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on any of the clinical chemistry parameters of the male and female rats previously treated with the test item for 90 days compared to the control group at the end of the recovery period on test day 119.

However, statistically significant differences in clinical chemistry parameters compared to the control, which are not considered to be test item-related were found as follows:

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01) increase in bilirubin (total) was observed for male rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

ENDOCRINE FINDINGS
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on any of the thyroid hormone (T3/T4) levels of the male and female rats treated with the test item for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg/bw/day: no test item-related influence was noted on any of the thyroid hormone (T3/T4) levels of the male and female rats previously treated with the test item for 90 days compared to the control group at the end of the recovery period on test day 119.

However, the mean TSH-serum level of the female animals treated with 300 or
1000 mg/kg bw/day appeared to be reduced by 13% or 84% compared to the control group (statistically significant at p ≤ 0.01 for the high dose females). Male animals were not affected in a similar way. The effect at the intermediate dose was marginal, but it must be considered that the serum TSH level was below the lowest level of quantification (= 0.100 ng/mL) for 7/10 females. At the high dose level, the serum TSH level of 8/10 females was
No test item-related influence was noted for the serum levels of T3 and T4. At the end of the recovery period on test day 119, the TSH-serum level of the female rats previously treated with 1000 mg/kg bw/day via the diet for 90 days was still decreased by 38% compared to the control group. The serum TSH level was still below the lowest level of quantification (= 0.100 ng/mL) for 3/5 females.

The above finding can be put into perspective because of the following
considerations: in the present study, no changes whatsoever were noted in the
thyroid weight and the histopathology of the thyroids. Thus, the decreased TSH
levels that were noted for the animals were judged not to be of toxicological
relevance and were not considered in the derivation of the NOAEL. This
assessment is in accordance with a publication by Siglin, J.C. et al., 2000*: dose
levels with significant changes for TSH levels, but without changes in thyroid
hormones, thyroid weight and histopathology or other indicators of toxicity (body weight, adverse clinical signs) are not considered for determining the NOAEL.

It is generally known that histopathological examination of the thyroid is usually
more sensitive than thyroid weight and hormone levels (Beekhuijzen M. et al.
(2016)*. The validation report of OECD 407 (OECD, 2006)* states that 'Thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weight was reliable, but somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, and the standard operating procedures for blood sampling and for thyroid hormone analyses are not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values.'

However, statistically significant differences in thyroid hormone levels compared to the control, which are not considered to be test item-related were found as follows:

- 100 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in thyroid hormone T3 was observed for female rats on test day 91. This findings lacks dose-dependency.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01) decrease in TSH was observed for female rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

*References:
Siglin, J. C. et al. (2000). A 90-Day Drinking Water Toxicity Study in Rats of the
Environmental Contaminant Ammonium Perchlorate. Toxicological Sciences 57. 61 - 74.

Beekhuijzen M. et al. (2016). Update of OECD DART guidelines with endocrine
disruptor relevant endpoints: practical considerations. Reproductive Toxicology 64. 64-71.

OECD (2006). Report of the Validation of the Updated Test Guideline 407: Repeat
Dose 28-day Oral Toxicity Study in Laboratory Rats. Series on Testing and
Assessment No 59, ENV/JM/MONO(2006)26.

URINALYSIS FINDINGS
Main study and recovery groups
Treatment and recovery period:
-100, 300 and 1000 mg/kg bw/day: no test item-related changes in the urinary status were noted for the male and female rats treated with the test item for 90 days compared to the control group at the end of the treatment period on test day 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on the urinary status of the male and female rats previously treated with the test item for 90 days compared to the control group at the end of the recovery period on test day 119.

However, statistically significant differences in urine parameters compared to the control, which are not considered to be test item-related were found as follows:

- 300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in relative volume was observed for female rats on test day 91. The slight alteration in comparison to control animals is without any biological relevance

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in specific gravity was observed for female rats on test day 119. Furthermore, a statistically significant (p ≤ 0.01 or p ≤ 0.05) decrease in pH and relative volume was observed for female rats on test days 91 (relative volume only) and 119. The slight alterations in comparison to control animals are without any biological relevance.

Please also refer to the field "Attached background material".

BEHAVIOUR (FUNCTIONAL FINDINGS)
Main study and recovery groups
Treatment period and recovery period:
- 100, 300 and 1000 mg/kg bw/day: the neurological screening performed at the end of the treatment period in test week 13 (main study) did not reveal any test item-related influence in the male and female rats treated with the test item for 90 days, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.

- 1000 mg/kg bw/day: no test item-related influence was noted on the parameters of the neurological screening of the male and female animals previously treated with the test item for 90 days at the end of the 4-week recovery period.

However, statistically significant differences in neurological parameters of main study and recovery animals compared to the control, which are not considered to be test item-related were found as follows:

Main study:
- 100 mg/kg bw/day: a statistically significant (p ≤ 0.01) decrease in forelimb grip strength was observed for female rats in test week 13. The slight alteration in comparison to control animals is without any biological relevance.

- 300 mg/kg bw/day: a statistically significant (p ≤ 0.01) decrease in forelimb grip strength was observed for female rats in test week 13. The slight alteration in comparison to control animals is without any biological relevance.

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01 or p ≤ 0.05) decrease in hind leg splay and hindlimb grip strength was observed for male rats in test week 13. The slight alteration in comparison to control animals is without any biological relevance

Recovery group:
- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in hindlimb grip strength was observed for male rats in test week 17. The slight alteration in comparison to control animals is without any biological relevance.

Please also refer to the field "Attached background material".

ORGAN WEIGHTS
1) Main study and recovery animals
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative and absolute organ weights of the male and female animals treated with the test item for 90 days compared to the control group at the end of the treatment period on test days 91.

- 1000 mg/kg bw/day: no test item-related influence was noted on the relative and absolute organ weights of the male and female animals previously treated with the test item for 90 days at recovery sacrifice on test day 119.

However, statistically significant differences in relative and absolute organ weights compared to the control, which are not considered to be test item-related were found as follows:

Absolute organ weights:
- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in left testis weight as well as left and right ovary weight was observed for male and female rats, respectively, on test day 119. The slight alteration in comparison to control animals is without any biological relevance.

Relative organ weights:
- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in pituitary weight as well as left and right ovary weight was observed for female rats on test days 91 (pituitary weight only) and 119. The slight alteration in comparison to control animals is without any biological relevance.

2) Satellite groups:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the relative and absolute liver and spleen weights of the male and female animals treated with the test item for 91 days compared to the control group at the end of the treatment period on test days 92.

Please also refer to the field "Attached background material".

GROSS PATHOLOGICAL FINDINGS
1) Main study and recovery animals
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: macroscopic inspection at necropsy did not reveal any test item-related changes in the organs or tissues of the animals treated with the test item for 90 days at terminal sacrifice on test day 91.

- 1000 mg/kg bw/day: no changes were noted in the organs or tissues of the animals previously treated with the test item for 90 days at recovery sacrifice on test day 119.

A small number of macroscopic findings such as changes in the stomach mucosa (dark-red focus) or pituitary (enlarged) were noted in individual animals of the test item-treated groups and are considered to be incidental findings.

2) Satellite animals
Treatment period:
- 100, 300 and 1000 mg/kg bw/day: macroscopic inspection at necropsy did not reveal any test item-related changes in the organs or tissues of the animals treated with the test item for 91 days at terminal sacrifice on test day 92.

A macroscopic finding in form of pyelectasia in the kidney was noted in few
individual animals only of the test item-treated groups and was considered to be
an incidental finding.

HISTOPATHOLOGICAL FINDINGS- NON-NEOPLASTIC
Main study and recovery groups
Treatment and recovery group:
- 0, 100, 300 and 1000 mg/kg bw/day: daily exposure to a diet containing the test item for a period of 90 days did not result in any test item-related gross findings or histopathological findings in a broad range of organs and tissues.

AUDITORY EXAMINATION
Main study and recovery groups
Treatment and recovery period:
- 100, 300 and 1000 mg/kg bw/day: there was no indication of any impairment to auditory acuity.

IRON LEVEL ANALYSIS (main group and satellite animals)
Plasma samples:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the iron plasma levels of the male and female rats treated with the test substance for 91 days compared to the control group at the end of the treatment period. No statistically significant differences were noted compared to the control group. The variations seen are a reflection of the physiological circadian variation of iron plasma levels.

Spleen samples:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the iron concentrations in spleen samples of the male and female rats treated with the test item for 91 days compared to the control group at the end of the treatment period. No statistically significant differences were noted compared to the control group.

Liver samples:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence was noted on the iron concentrations in liver samples of the male and female rats treated with the test item for 91 days compared to the control group at the end of the treatment period. No statistically significant differences were noted compared to the control group.

QUANTIFICATION OF ABERRANT CRYPT FOCI (non-GLP; all main study and recovery animals of the control group and high dose group)

Visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
In the current repeated dose toxicity study, iron oxide Sicovit® Yellow 10 E172 was administered via diet to groups of 10 male and 10 female Crl:CD (SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred daily for a 90- day period. A control group receiving plain diet was run concurrently. Furthermore, recovery groups (n = 10 animals/sex/group; control group and high dose level only; recovery period: 4 weeks) and a satellite group (n = 10 animals/sex/group: treatment period: 91 days) were also run concurrently.

During the observation of animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, haematological findings, clinical chemistry findings, endocrinological findings, urinalysis findings, behaviour (functional findings), organ weights, gross pathology and histopathological findings. Futhermore, iron level analysis of plasma and tissues (spleen and liver) revealed no test item-related influence on iron concentration in plasma and spleen samples. Lastly, visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species.

Based on the findings, the no observed adverse effect level (NOAEL) for general toxicity is considered to be greater than 1000 mg/kg bw/day for male and female rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study - very well documented, study was performed with Fe3O4 as a representative for the iron oxide group - see Category approach for Fe2O3, Fe3O4, FeOOH, ZnFe2O4 and (Fe,Mn)2O3
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD was 1.3 µm, GDS ~2
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day 5 days/week
Remarks:
Doses / Concentrations:
4.7, 16.6, 52.1 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
20
Control animals:
yes
Dose descriptor:
NOAEL
Effect level:
4.7 mg/m³ air
Sex:
male/female
Basis for effect level:
other: see "Remarks"
Critical effects observed:
not specified
Rats exposed subchronically to three different concentrations of Fe3O4 revealed findings clearly consistent with and typical for a poorly  soluble particle. Congruent with previous studies addressing the  retention kinetics of inhaled Fe3O4 particles, neither analytical nor  toxicological evidence existed that free, biosoluble iron was liberated  from the inhaled dust to any appreciable extend. Also in this study no  evidence of extrapulmonary toxicity existed. With regard to indices  considered to be adverse, viz. increased counts of cells and especially  PMNs in BAL, elevated LDH as marker of cytotoxicity, and ß-NAG as marker of increased lysosomal activities 4.7 mg/m³ constitute an exposure level  without evidence of adversity. These findings match those observed by  histopathology.
Executive summary:
Rats exposed subchronically to three different concentrations of Fe3O4 revealed findings clearly consistent with and typical for a poorly soluble particle. The retention kinetics of inhaled Fe3O4 particles revealed neither analytical nor toxicological evidence that free, biosoluble iron was liberated from the inhaled dust to any appreciable extend. Also in this study no evidence of extrapulmonary toxicity existed. The results of this study support the view, that the NOAEL of Fe3O4 is 4.7 mg/m³.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Quality of whole database:
GLP guideline study according OECD #413 (Subchronic Inhalation Toxicity: 90-Day)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study - very well documented, study was performed with Fe3O4 as a representative for the iron oxide group - see Category approach for Fe2O3, Fe3O4, FeOOH, ZnFe2O4 and (Fe,Mn)2O3
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD was 1.3 µm, GDS ~2
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day 5 days/week
Remarks:
Doses / Concentrations:
4.7, 16.6, 52.1 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
20
Control animals:
yes
Dose descriptor:
NOAEL
Effect level:
4.7 mg/m³ air
Sex:
male/female
Basis for effect level:
other: see "Remarks"
Critical effects observed:
not specified
Rats exposed subchronically to three different concentrations of Fe3O4 revealed findings clearly consistent with and typical for a poorly  soluble particle. Congruent with previous studies addressing the  retention kinetics of inhaled Fe3O4 particles, neither analytical nor  toxicological evidence existed that free, biosoluble iron was liberated  from the inhaled dust to any appreciable extend. Also in this study no  evidence of extrapulmonary toxicity existed. With regard to indices  considered to be adverse, viz. increased counts of cells and especially  PMNs in BAL, elevated LDH as marker of cytotoxicity, and ß-NAG as marker of increased lysosomal activities 4.7 mg/m³ constitute an exposure level  without evidence of adversity. These findings match those observed by  histopathology.
Executive summary:
Rats exposed subchronically to three different concentrations of Fe3O4 revealed findings clearly consistent with and typical for a poorly soluble particle. The retention kinetics of inhaled Fe3O4 particles revealed neither analytical nor toxicological evidence that free, biosoluble iron was liberated from the inhaled dust to any appreciable extend. Also in this study no evidence of extrapulmonary toxicity existed. The results of this study support the view, that the NOAEL of Fe3O4 is 4.7 mg/m³.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
4.7 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP guideline study according OECD #413 (Subchronic Inhalation Toxicity: 90-Day)

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

Introductory remark on read-across:


In this dossier, the endpoint repeated dose toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the iron oxide category. The predominant characteristic of the iron oxide category substances is the inertness being a cause of their chemical stability and very poor reactivity. This is shown by a very low dissolution in water and artificial physiological fluids as well as a very low in vivo bioavailability after oral administration. This very low reactivity, solubility and bioavailability leads to a complete lack of systemic toxicity after acute, sub-acute or sub-chronic oral or inhalation exposure up to the limit dose of the maximum tolerated concentration of the respective test. Similarly, a lack of systemic effects for the endpoints mutagenicity and reproductive toxicity are anticipated. Further information on the read-across approach is given in the report attached to IUCLID section 13.2.


 


Inhalation:


In a subacute inhalation toxicity study, 48 male rats per group were exposed to three different aerosolized iron oxide powders (Fe2O3, Fe3O4 and FeOOH). Exposure was 6-hours/day on five days/week for two consecutive weeks. The rats were exposed to mean actual concentrations of 185.6 mg/m³ Fe3O4, 210.2 mg/m³ Fe2O3 and 195.7 mg/m³ FeOOH (Pauluhn, 2005). The repeated exposure to the aerosolized iron oxides was not associated with any specific clinical signs, changes in body temperature or body weights. Histopathological evaluation of rat lungs exposed to the different iron oxides revealed findings consistent with a 'poorly soluble particle' effect after the 2-week exposure period, including the 3-month post-exposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs was not observed (Pauluhn, 2005). This supports the conclusion, that Fe3O4 can serve as a surrogate for FeOOH and Fe2O3.


For Fe3O4 valid subacute and subchronic inhalation studies are available (Pauluhn, 2006a; Pauluhn, 2006b). In the subacute inhalation toxicity study 30 male rats were exposed to 10.1, 19.4, 45.6 and 95.8 mg/m³ Fe3O4 for 6 hours/day, 5 days/week for 4 weeks and serially sacrificed 1, 8, 24 weeks after the 4 weeks exposure period. Clinical signs were recorded daily before and after exposure or once per week during the post-exposure period. At each serial sacrifice, inflammatory endpoints were determined in bronchoalveolar lavage (BAL). Rats were subjected to gross pathological examination and histopathology (nasal passages, trachea, lung, liver, spleen, kidneys, testes and thymus). The repeated 4-week exposure to the aerosolized dry powder was not associated with specific clinical signs or consistent changes in body weights. Changes in organ weights occurred and consisted of increased lung and lung-associated lymph nodes (LALN) weight at 45.6 mg/m³ and above. Histopathological evaluation of rat lungs exposed to Fe3O4 revealed finding consistent with a poorly soluble particle effect. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs to any appreciable extent was not found. Extrapulmonary effects causally linked to the exposure of Fe3O4 were not found at any exposure concentration and time point (Pauluhn, 2006b). In the subchronic inhalation toxicity study in rats (20 male and 20 female rats per group) the animals were exposed 6 hours/day, 5 days/week for 13 weeks to 4.7, 16.6 and 52.1 mg/m³ Fe3O4. During the study, the body weights were determined twice weekly and clinical signs were recorded daily before and after exposure. At sacrifice, inflammatory endpoints were determined in BAL. Histopathology focused on the entire respiratory tract (nasal passages, trachea, lung, lung associated lymph nodes) and included all extrapulmonary organs as suggested by OECD 413. At sacrifice biological specimens were collected for hematology, clinical pathology and urinalysis (Pauluhn, 2006a). The repeated exposure of rats during a study period of 13 weeks was not associated with any specific clinical signs. Hematology, clinical pathology and urinalysis were unobtrusive. Neither analytical nor toxicological evidence existed that free, biosoluble iron was liberated from the inhaled dust to any appreciable extent. However, the neutrophils in male rats and some biochemical markers were elevated at 4.7 mg/m³ and above (Pauluhn, 2006a). The NOAECs for Fe3O4 are 10.1 mg/m³ for the subacute exposure and 4.7 mg/m³ for subchronic exposure in rats (Pauluhn, 2006a; Pauluhn, 2006b).


A histopathological evaluation of rat lungs exposed to three different iron oxides revealed findings consistent with a 'poorly soluble particle' effect after the 2- week exposure period, including the 3-month post-exposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxides were not be detected at any time point. At the end of the 3-month post-exposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchioloalveolar hypercellularity) showed a decrease in incidence and/or severity. The comparative assessment of the three different iron oxides revealed the same quality and time course of responses, i.e., marked differences of any toxicological significance between the test specimens were not observed. Nonetheless, there was a consistent trend that the rats exposed to Fe3O4 displayed minimally more pronounced changes when compared to Fe2O3 and FeOOH. This supports the conclusion that from a scientific point of view further testing of Fe2O3 and Fe3O4 does not appear to be justified and that future studies with Fe3O4 can serve as conservative proxy for Fe2O3 and FeOOH (Pauluhn, 2005).


In a short-term (5-day) inhalation study (BASF SE 2015), 8 male Wistar rats (5 animals in the main group and 3 animals in the recovery group) were exposed to 10 and 30 mg/m³ of smaller nano-sized Fe2O3 and 30 mg/m³ of larger nano-sized Fe2O3. Dust Inhalation exposure of rats to 10 and 30 mg/m³ test item 1 and 30 mg/m³ of test item 2 on 5 consecutive days did not cause any adverse effect in the respiratory tract, which was examined by broncho-alveolar lavage and histopathology. There were no changes of hematology and clinical chemistry parameters. A slightly decreased mean body weight and intermittently reduced body weight change were observed during exposure and post-exposure period of the test group with micro-sized Fe2O3. However, the deviations to the control group were not biologically relevant. Thus, under current study conditions, the NOAEC was 30 mg/m³ for both nano-sized samples. No relevant differences between the smaller nano-sized and the larger nano-sized Fe2O3 were observed.


In consideration of the results of the repeated dose inhalation studies with ZnO and the repeated dose inhalation studies in rats and monkeys with Mn3O4 it can be concluded, that the NOEC of Fe2O3, FeOOH, ZnFe2O4, (Fe,Mn)2O3, and (Fe,Mn)3O4 is in the same range as for Fe3O4, both for nano-sized and micro-sized particles (see Category approach justification, IUCLID section 13.2).


 


Oral:


In the repeated dose toxicity study by Hansen (2020, unpublished report), FeOOH (Iron oxide Sicovit® Yellow 10 E172) was administered via diet to groups of 10 male and 10 female Crl:CD (SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred daily for a 90-day period. A control group receiving plain diet was run concurrently. Furthermore, recovery groups (n = 10 animals/sex/group; control group and high dose level only; recovery period: 4 weeks) and a satellite group (n = 10 animals/sex/group: treatment period: 91 days) were also run concurrently. During the observation of animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, haematological findings, clinical chemistry findings, endocrinological findings, urinalysis findings, behaviour (functional findings), organ weights, gross pathology and histopathological findings. Furthermore, iron level analysis of plasma and tissues (spleen and liver) revealed no test item-related influence on iron concentration in plasma and spleen samples. Lastly, visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species. Based on the findings, the no observed adverse effect level (NOAEL) for general toxicity is greater than 1000 mg/kg bw/day for male and female rats.


 


In the repeated dose toxicity study by Hansen (2020, unpublished report), Fe3O4 (Ferroxide® Black 86) was administered via diet to groups of 10 male and 10 female Crl:CD (SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred daily for a 90- day period. A control group without treatment was run concurrently. Recovery groups (n = 10 animals/sex/group; control group and high dose level only; recovery period: 4 weeks) and a satellite group (n = 10 animals/sex/group: treatment period: 91 days) were also run concurrently. During the observation of animals, no test item-related effects were observed in animals for clinical signs, mortality, body weight and weight changes, food consumption, water consumption, ophthalmological findings, haematological findings, clinical chemistry findings, endorcinoloigcal findings, urinalysis findings, behaviour (functional findings), organ weights, gross pathology and histopathological findings. Furthermore, iron level analysis of plasma and tissues (spleen and liver) revealed no test item-related influence on iron concentration in plasma and spleen samples. However, the iron concentrations in liver tissue samples were slightly increased by 35% to 63% for the female animals treated with 100 or 300 mg/kg bw/day of the test item via the diet and for the male and female animals treated with 1000 mg/kg bw/day via the diet for 91 days compared to the control group at the end of the treatment period. All values were statistically significant at p ≤ 0.01 compared to the control group. However, no dose-response relationship was noted. Lastly, visual inspection by microscopy of the complete colon of all submitted rats did not identify aberrant crypt foci (ACF) in any of the submitted tissue samples. Based on the results of this analysis it can be concluded that daily oral administration of the test item to rats up to a daily dose of 1000 mg/kg bw and over a time period of 90 days via diet did not induce ACF in the colon of this species. Based on the lack of any adverse findings, the no observed adverse effect level (NOAEL) for general toxicity is greater than 1000 mg/kg bw/day for male and female rats.


 


A subchronic toxicity study of various orally administered nanoparticles including red iron oxide (Fe2O3, 60-118 nm) was performed by Yun et al. (2015) according to the OECD Test Guideline (TG) 408 (OECD, 1998). Sprague-Dawley rats received daily doses of 250, 500 or 1000 mg/kg bw/day for 13 weeks by gavage. Fe2O3 nanoparticles had no significant effects on body weight, mean daily food and water consumption, when compared with control groups. There were no treatment-related changes in haematological, serum biochemical parameters or histopathological observations. Some changes in organ weights were observed: decreases in weight of pituitary gland and liver and increases in weight of adrenal gland and testis. According to the authors, ‘these changes were sporadic without dose-dependent trends, indicating that they were not considered toxicologically relevant’. In blood and all tissues tested, including liver, kidney, spleen, lung and brain, the concentration of Fe showed no dose-associated response in comparison to the control groups. Iron concentrations in the urine of Fe2O3 nanoparticle-treated rats showed no significant differences compared to those of control animals. Although not statistically significant, the concentrations of Fe in the faeces of treated animals were found to be higher than those of the control groups. The authors stated that the subchronic oral dosing with Fe2O3 nanoparticles showed no systemic toxicity to rats. The NOAEL was established at 1000 mg/kg bw/day, the highest dose tested in rats receiving Fe2O3 nanoparticles by gavage.


 


 


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:


 


In a comparative subacute inhalation toxicity in rats with the iron oxides (FeOOH, Fe2O3 and Fe3O4 also named as  'Black', 'Yellow', 'Red') no significant differences in histopathology and clinical signs were found. Therefore repeated dose studies with longer duration (4 or 13 weeks) were conducted with Fe3O4 as a surrogate for iron manganese oxide - ( see Category approach for Fe2O3, Fe3O4, FeOOH, (Fe,Mn)2O3, (Fe,Mn)3O4, and ZnFe2O4.


 


 


Justification for selection of repeated dose toxicity inhalation - local effects endpoint:


 


In a comparative subacute inhalation toxicity in rats with the iron oxides (FeOOH, Fe2O3 and Fe3O4 also named as  'Black', 'Yellow', 'Red') no significant differences in histopathology and clinical signs were found. Therefore repeated dose studies with longer duration (4 or 13 weeks) were conducted with Fe3O4 as a surrogate for iron manganese oxide - see Category approach for Fe2O3, Fe3O4, FeOOH, (Fe,Mn)2O3, (Fe,Mn)3O4, and ZnFe2O4.

Justification for classification or non-classification

In summary, the members of the iron oxide category are inert particles, therefore no toxicological effects are expected for repeated oral and dermal toxicity. The NOAECs for Fe3O4 are 10.1 mg/m³ for the subacute exposure and 4.7 mg/m³ for subchronic exposure in rats.


A classification is therefore not justified.