Mill scale (ferrous metal)

Endpoint summary

Description of key information

Repeated dose toxicity -  oral:

The iron oxides, show a complete absence of adverse effects in guideline compliant repeated dose toxicity studies via the oral route. Neither macroscopic, nor microscopic findings were noted. These findings were seen in a sub-chronic oral study in rats with three different iron oxides. An NOAEL was not derived based on the absence of adverse effects up to the limit dose of 1000 mg/kg bw/day.

A number of sub-chronic oral repeated dose toxicity studies with iron show a substantial variability in the effect doses (NOAEL range from 68 to 1300 mg Fe/kg bw/day, with a mean of 390 mg Fe/kg bw/day). The effects described were mild increase in relative liver weight and increase in liver non-haeme Fe. In a double-blind clinical trial some very mild effects are described after treatment of anaemic patients with iron (powder) as food supplement at 1800 mg Fe/day for three weeks. The treatment was generally well-tolerated with transient gastrointestinal side effects comparable to the effects seen with soluble iron salts. A comparative group of patients was treated with a soluble iron salt at 180 mg Fe/day, very similar effects were observed compared with the iron-treated group. This data shows an approx. 10-fold lower toxicity for iron compared with soluble iron salts.

For comparative purposes, toxicological data on soluble iron salts were included in this dossier The sub-acute animal studies with soluble iron salts (e.g. iron sulfate, iron chloride and iron tartrate) indicate an effect dose of 60-180 mg Fe/kg bw/day based on increased liver weights and inflammatory parameters in the gastro-intestinal tract. The effects in the gastro-intestinal tract are also identified as lead-effect in the human studies, where effect levels of 60-200 mg Fe are described.

The lower effect levels for highly soluble and highly bioavailable iron substances compared to the higher effect level for iron for both in animals and humans, corroborates the assumption that solubility in water or artificial body fluids correlates with the systemic toxicity of the iron category substances.

 

Repeated dose toxicity - inhalation:

The existing 28-day and 90-day inhalation studies with iron oxides in rats does not show any substance-related adverse effects. The effects of iron oxides after 28-day and 90-day inhalation is best compared with the effects seen with other poorly-soluble low-toxicity particles (PSLT), leading to a minimal or mild inflammatory response only at the maximum tolerated concentration in repeated dose toxicity studies via inhalation. Furthermore, in human epidemiological studies following prolonged inhalation exposure, no clinically significant adverse local or systemic effects were reported.

Based on the physico-chemical properties of the particles, direct systemic exposure to iron upon inhalation can be ruled out. Based on the local effects particles can have in the respiratory tract and the physico-chemical properties of carbonyl iron, it is not expected that a longer duration would result in the detection of other effects, not solely determined by the PSP character of carbonyl iron.

 

Repeated dose toxicity – dermal:

There is no information available on dermal toxicity. The conduct of repeated dose toxicity study is not considered to be required since inhalation of the substance is considered the most relevant route of human exposure. Furthermore, physicochemical and toxicological properties of the iron category substances do not suggest a significant rate of absorption through the skin (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

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

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 (control group: 255.12 ± 18.40 g; treatment group: 274.63 ± 10.23 g). However, the stated body weight findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). 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 (control group: 300.24 ± 30.37 g; treatment group: 331.91 ± 27.99 g). However, the stated body weight findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). 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 food consumption of male rats was observed in test weeks 10, 11 and 12. However, the stated food consumption findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group
Week 10: 49.43 ± 1.84 g/kg bw/day
Week 11: 48.52 ± 2.04 g/kg bw/day
Week 12: 47.75 ± 2.01 g/kg bw/day
Treatment group
Week 10: 51.76 ± 2.46 g/kg bw/day
Week 11: 50.99 ± 3.07 g/kg bw/day
Week 12: 50.07 ± 2.41 g/kg bw/day

- 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 6, 9, 10, 11 and 12. However, the stated food consumption findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group
Week 6: 55.12± 2.57 g/kg bw/day
Week 9: 50.71 ± 1.69 g/kg bw/day
Week 10: 49.43 ± 1.84 g/kg bw/day
Week 11: 48.52 ± 2.04 g/kg bw/day
Week 12: 47.75 ± 2.01 g/kg bw/day
Treatment group
Week 6: 57.60 ± 2.24 g/kg bw/day
Week 9: 52.85 ± 2.46 g/kg bw/day
Week 10: 51.75 ± 2.04 g/kg bw/day
Week 11: 51.20 ± 2.29 g/kg bw/day
Week 12: 50.31 ± 2.36 g/kg bw/day

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 food consumption of female rats was observed in test week 12. However, the stated food consumption findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group:
Week 12: 58.67 ± 3.55 g/kg bw/day
Treatment group
Week 12: 64.05 ± 3.44 g/kg bw/day

-300 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in food consumption of male rats was observed in test week 5. The slight alteration in comparison to control animals is without any biological relevance.
Control group:
Week 5: 56.54 ± 2.13 g/kg bw/day
Treatment group
Week 5: 53.69 ± 2.88 g/kg bw/day

-1000 mg/kg bw/day: a statistically significant (p ≤ 0.01) decrease in body weight of male rats was observed in test week 7. However, the stated food consumption findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group:
Week 7: 57.57 ± 2.01 g/kg bw/day
Treatment group
Week 7: 54.07 ± 2.48 g/kg bw/day

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. However, the stated haematological finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group (x10E3/µL)
Test day 91: 0.017 ± 0.005
Treatment group
Test day 91: 0.011 ± 0.003

- 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. However, the stated haematological finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance and dose-dependency was lacking.
Control group (x10E3/µL)
Test day 91: 0.049 ± 0.012
Treatment group
Test day 91: 0.073 ± 0.026

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) and alkaline phosphatase (females) were observed on test day 120. However, the stated clinical chemistry findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.

Protein (males)
Control group (test day 120): 60.0 ± 1.2 g/L
Treatment group (test day 120): 63.0 ± 1.9 g/L

Alkaline phosphatase (females)
Control group (test day 120): 32.2 ± 7.2 U/L
Treatment group (test day 120): 44.4 ± 6.5 U/L

- 1000 mg/kg bw/day: statistically significant (p ≤ 0.05) increases in sodium (males) was observed on test day 120. The slight alteration in comparison to control animals is without any biological relevance.
Sodium (males)
Control group (test day 120): 142.0 ± 0.7 mmol/L
Treatment group (test day 120): 143.2 ± 0.8 mmol/L


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.
Control group (test day 120; males): 34.4224 ± 3.991 nmol/L
Treatment group (test day 120; males): 42.6472 ± 5.8714 nmol/L

Control group (test day 91; females): 25.0712 ± 1.8352 nmol/L
Treatment group (test day 91; females): 30.2174 ± 5.7863 nmol/L

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. However, the stated urinalysis findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below).The slight alteration in comparison to control animals is without any biological relevance.
Control group:
Test day 91: 6.17 ± 0.24
Test day 120: 6.66 ± 0.56
Treatment group:
Test day 91: 6.55 ± 0.40
Test day 120: 6.20 ± 0.07

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.

Hindleg splay (females)
Control group (test week 13): 8.0 ± 1.1 cm
Treatment group (test week 13): 8.2 ± 0.9 cm

Forelimb grip strength (males)
Control group (test week 13): 297.5 ± 46.0 g
Treatment group (test week 13): 255.3 ± 49.9 g

Hindlimb grip strength (males)
Control group (test week 13): 54.4 ± 15.4 g
Treatment group (test week 13): 66.2 ± 9.9 g

- 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. Furthermore, the stated findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below).
Control group (number of movements/12 min):
Test week 13: 106.6 ± 26.3
Treatment group (number of movements/12 min):
Test week 13: 131.0 ± 71.5

Recovery groups:
- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) decrease in hindlimb grip strength was observed for female rats in test week 17. The slight alteration in comparison to control animals is without any biological relevance.
Hindlimb grip strength (females)
Control group (test week 17): 72.1 ± 21.5 g
Treatment group (test week 17): 40.0 ± 16.3 g

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. However, the stated finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance and lacking dose-dependency.
Control group (test week 91): 3.937 ± 0.332 g/kg bw
Treatment group (test week 91): 3.540 ± 0.212 g/kg bw

- 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. Furthermore, the stated findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below).

300 mg/kg bw/day:
Control group (test wek 91): 0.0189 ± 0.0060 g/kg bw
Treatment group (test week 91): 0.0284 ± 0.0051 g/kg bw

1000 mg/kg bw/day:
Control group (test wek 91): 0.0189 ± 0.0060 g/kg bw
Treatment group (test week 91): 0.0277 ± 0.0094 g/kg bw

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.05) increase in liver weight was observed for female rats in test week 120. However, the stated finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group (test week 120): 26.45 ± 1.71 g/kg bw
Treatment group (test week 120): 29.34 ± 1.78 g/kg bw

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. However, the stated finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance and lacking dose-dependency.
Control group (test week 91): 1.106 ± 0.087 g
Treatment group (test week 91): 0.995 ± 0.083 g

- 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. Furthermore, the stated findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below)

300 mg/kg bw/day:
Control group (test week 91): 0.0094 ± 0.003 g
Treatment group (test week 91): 0.0143 ± 0.0028 g

1000 mg/kg bw/day:
Control group (test week 91): 0.0094 ± 0.003 g
Treatment group (test week 91): 0.0135 ± 0.0042 g


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. However, the stated finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). Dose-dependency was lacking.
Control group (test week 92): 1.558 ± 0.243 g/kg bw
Treatment group (test week 92): 1.794 ± 0.164 g/kg

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.
Control group (test week 92): 0.809 ± 0.126 g
Treatment group (test week 92): 0.961 ± 0.093 g/kg

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.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproductive/developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001-08-13 to 2001-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996-03-22

Deviations:

yes

Remarks:

Males not dosed during mating. Length of mating period not clearly stated. Detailed clinical observations & neurobehaviour investigation missing. Runts not recorded. Potassium & bile acids were not measured. Historical control data missing.

GLP compliance:

yes

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 at room temperature, sealed under argon gas
- Stability under test conditions: test substance was stable throughout its period of use

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This species (rat) is commonly used for toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Crj: CD(SD) IGS, SPF
- Source: Charles River Laboratories (Hino Breeding Center)
- Age at start of administration: 10 weeks old
- Weight at start of administration: males: 341 - 383 g; females: 222 - 255 g
- Fasting period before administration: yes
- Housing:
Quarantine and acclimatisation period: stainless steel suspended cages (240 mm wide, 380 mm deep, 200 mm high), five animals per cage;
After group allocation: individually in stainless steel five-chamber cages (755 mm wide, 210 mm deep, 170 mm high).
Mating: stainless steel suspended cages.
From day 18 of gestation: dams were reared individually in plastic cages (310 mm wide, 360 mm deep, 175 mm high) provided with autoclaved bedding

- Diet (ad libitum): solid food (CRF-1, Oriental Yeast Co.)
- Water (ad libitum): tap water
- Quarantine period: 5 days
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: food and drinking water analysis results were all within the standard ranges.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 24 °C
- Humidity: 40 - 70 %
- Ventilation: 12/day
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

Oral administration was selected as the administration route because it is thought that the test item may be orally ingested by humans.

Vehicle:

other: water for injection

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in water for injection to a concentration of 200 mg/mL. The resulting 200 mg/mL solution was serially diluted using water for injection to concentrations of 60, 20 and 6 mg/mL. The calculations were performed according to the purity when the test substance was prepared.
The prepared solutions of each concentration were prepared at the time of use, and used within six hours of preparation. Any administration sample remaining after administration was discarded.

DOSE VOLUME APPLIED:
- males: 5 mL/kg, calculated based on the body weights measured on dosing day or the lastest measurement.
- females: 5 mL/kg, calculated based on the body weights measured on dosing day or the lastest measurement before mating and during the mating period, and calculated based on the body weights measured on gestation days 0, 7, 14 and 21, and based on the body weights measured on lactation day 0.

VEHICLE
- Lot no.: 0H93N

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

It was verified that there were no problems with the stability of 2-200 mg/mL prepared solutions that had been kept for six hours at room temperature, shielded from light (Watanabe)*.

The test substance concentration in each administration sample used was measured by the titration method on the first day of administration of the males and on the last day of administration of the females. The results revealed that the test substance concentrations were 99.9-108.0 % of the concentrations displayed.

Reference:
- Watanabe T, et al: Iron II sulfate heptahydrate stability verification study (Study no 093320) (Hashima Laboratory, Nihon Bioresearch Inc.)

Duration of treatment / exposure:

males: 49 days (14 before mating and 35 days after mating)
females: 42 - 47 days (14 days before mating, throughout the mating period (max. 5 days), throughout the gestation period, and until lactation day 5)

Frequency of treatment:

daily

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

equivalent to 6 mg Fe/kg bw/day

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

equivalent to 20 mg Fe/kg bw/day

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

equivalent to 60 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 201 mg Fe/kg bw/day

No. of animals per sex per dose:

12 males / 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose was decided on considering the results of a preliminary study of oral administration for two weeks to male rats (0, 125, 250, 500 and 1000 mg/kg administered). Dark red discolouration of the glandular stomach mucosa was observed in the ≥ 250 mg/kg groups, salivation and thickening of the glandular stomach mucosa was observed in the ≥ 500 mg/kg groups, and decreased food consumption and a tendency to body weight decrease were observed in the 1000 mg/kg group. Therefore, in this study, the maximum dose was set at 1000 mg/kg and the lower doses were set at 300, 100 and 30 mg/kg.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before administration, and twice a day thereafter (once on the day of necropsy only)
- Cage side observations checked: general condition and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
males: twice a week (dosing days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, and 49 as well as on the day of necropsy)
females: twice a week throughout the pre-mating period and the mating period (dosing days 1, 4, 8, 11, 15, and 18) as well as on gestation days 0, 7, 14, and 21, and on lactation days 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
males: food consumption was measured twice a week throughout the pre-mating period and until completion of the mating period (residual amount, measured on dosing days 3, 6, 10, 13, 24, 27, 31, 34, 38, 41, 45, and 48).
females food consumption was measured twice a week during the pre-mating period (residual amount, measured on dosing days 3, 6, 10 and 13) as well as on gestation days 2, 9, 16, and 21 and on lactation day 4.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day before final administration
- Anaesthetic used for blood collection: Yes, pentobarbital sodium (40 mg/kg)
- How many animals: 6 animals/sex/group (same animals as used for clinical chemistry and urinalysis)
- Parameters checked: erythrocyte count, haemoglobin level, heamatocrit level, platelet count, leucocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte content, differntial count, prothrombin time, activated partial thromboplastin time and fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day before final administration
- How many animals: 6 animals/sex/group (same animals as used for haematology and urinalysis)
- Parameters checked: AST, ALT, ALP, γ-GLP, total protein, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglycerides, calcium, inorganic phosphorus, iron, sodium, phosphorous, chloride, albumin level, protein fraction, and albumin/globulin.

URINALYSIS: Yes
- Time schedule for collection of urine: before completion of the administration period, urine samples were collected after a fasting period (fresh urine) and during a 24 hour period (food restored)
- Urine-sampling-cages used for collection of urine: Yes
- Animals fasted: Yes, but given water
- How many animals: 6 animals/sex/group (females had given birth)
- Parameters checked:
1) fresh urine (sampled before administration): colour, external appearance, pH, protein, glucose, ketone bodies, bilirubin, occult blood and urobilinogen and urinary sediment
2) 24 hour urine: urine volume (calculated from specific gravity and weight), and specific gravity

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Any animals found to have died were necropsied promptly.
All rats were sacrificed at the end of the study. The day after the final administration (dosing day 50 for the males, lactation day 6 for the females), the animals were anaesthetised, exsanguinated, and necropsied. The weights of the brain (cerebrum, cerebellum, medulla), pituitary, thyroid, thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymis, ovaries and uterus were measured. The relative organ weights of all organs weighed were calculated. The lungs, pituitary, thyroid, trachea, pancreas, salivary glands (sublingual and submandibular), oesophagus, stomach, duodenum, coelenteron, ileum, caecum, colon, rectum, lymph nodes (submandibular, mesenteric), bladder, seminal vesicles, testes, edpididymis, eyeballs, prostate, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, harderian gland, bone marrow (sternal, femoral), bones (sternum, femur) and mammary glands (females only) were fixed.
Twenty five days after mating, the females that did not give birth were exsanguinated under ether anaesthesia and necropsied. The number of corpora lutea and the number of implantations were counted. The brain (cerebrum, cerebellum, medulla), pituitary, thyroid, thymus, heart, liver, spleen, kidneys, adrenals, ovaries, lungs, trachea, pancreas, salivary glands (sublingual and submandibular), oesophagus, eyeballs, stomach, duodenum, coelenteron, ileum, caecum, colon, rectum, lymph nodes (submandibular, mesenteric), bladder, uterus, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, harderian gland, bone marrow (sternal, femoral), bones (sternum, femur) and mammary glands were fixed.

Paraffin-embedded specimens were prepared for the following organs and tissue harvested from the animals from which blood was sampled for haematology and blood biochemistry tests (6 animals/sex/group).
For the control group and 1000 mg/kg group (including the animals that died), histopathological examination was performed on the heart, lungs, trachea, liver, pancreas, salivary glands (sublingual and submandibular), oesophagus, stomach, duodenum, coelenteron, ileum, caecum, colon, rectum, thymus, spleen, lymph nodes (submandibular, mesenteric), kidneys, bladder, testes, epididymis, seminal vesicles, prostate, ovaries, uterus, vagina, pituitary, adrenals, thyroid, parathyroid (only if possible), brain (cerebrum, cerebellum, medulla), spinal cord, sciatic nerve, eyeballs, harderian gland, bone marrow (sternal, femoral), bones (sternum, femur) and mammary glands.
If the number of animals exhibiting abnormality in a particular tissue in the 1000 mg/kg group differed from the control group, the same histopathological examination was also performed for the same tissue from the animals in the 30, 100 and 300 mg/kg groups. The same histopathological examinations were also performed for the testes and epididymis, because one animal in the 1000 mg/kg group exhibited abnormality in the testes and epididymis on necropsy.
In the histopathological examination of the males and females in the 1000 mg/kg group, the liver tissue specimens were subjected to bile staining, iron staining and wear-and-tear pigment staining in order to identify the yellow-brown pigment observed in the liver.

Statistics:

Significant difference between the control group and each administration group was tested and displayed as p<0.05 and p<0.01. The post-copulation general condition, body weights and food consumption of the females that did not conceive were excluded from the respective totals.
Mean and standard deviation values for the following parameters were calculated for each group: body weight, food consumption, urine volume, urine specific gravity, haematology tests, blood biochemistry tests, and absolute and relative organ weights. Bartlett's test for homogeneity of variance was performed, and if there was homogeneity of variance, Dunnett's test was performed. If no homogeneity of variance was found, a Dunnett-type rank test was performed.
For tissue where effects indicative of toxicity were observed in the 1000 mg/kg group, the histopathological findings for the other dose groups were compared with the control group and between groups using the Dunnett-type rank test.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 1000 mg/kg group: body weights measured on dosing days 11 to 49 were significantly lower than those in the control group (p< 0.05 and p < 0.01).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 1000 mg/kg group: RBC (p<0.01), and APTT (p<0.01) were significantly lower than in the control group, and the MCV (p<0.01), MCH (p<0.01) and reticulocyte levels (p<0.05) were significantly higher than in the control group.

2) Females:
- 1000 mg/kg group: haemoglobin level was significantly higher than in the control group (p<0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 1000 mg/kg group: protein (p<0.01), albumin (p<0.01) and calcium levels (p<0.05) were significantly lower than in the control group, and the ALT, γ-GTP and A/G were significantly higher than in the control group (p<0.05).

2) Females:
- 300 mg/kg group: inorganic phosphorus levels were significantly higher than in the control group (p<0.05).
- 1000 mg/kg group: γ-GTP and inorganic phosphorus levels were significantly higher than in the control group (p<0.05).

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 1000 mg/kg group: absolute adrenal weights were significantly higher than in the control group (p<0.01) and the relative liver and adrenal weights were significantly higher than in the control group (p<0.01).

2) Females:
- 1000 mg/kg group: absolute and relative liver weights were significantly higher than in the control group (p<0.05 and p<0.01, respectively).

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1) Males (surviving)
1000 mg/kg/day:
- spleen: extramedullary haematopoiesis in four males, and yellow-brown pigment deposition in the red pulp in all six males. The extramedullary haematopoiesis observed was very mild to moderate. The yellow-brown pigment deposition in the red pulp was mild or moderate. It should be noted that the yellow-brown pigment deposition observed in the red pulp constituted a significant difference compared to the control group. For the extramedullary haematopoiesis observed in the 1000 mg/kg groups, the number of males affected and the severity differed compared to the control group.

300 mg/kg group:
- spleen: extramedullary haematopoiesis in five males, and yellow-brown pigment deposition in the red pulp in all six males. The extramedullary haematopoiesis observed was very mild or mild in the 300 mg/kg group. The yellow-brown pigment deposition in the red pulp was very mild or mild in the 300 mg/kg groups. For the extramedullary haematopoiesis observed in the 300 mg/kg group, the number of males affected and the severity differed compared to the control group.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS
1) Males:
- 0, 30, or 100 mg/kg group: no abnormalities in general condition were observed
- 300 mg/kg group: immediately after administration transient salivation was observed from dosing day 5 onwards (12/12 males)
- 1000 mg/kg group: immediately after administration transient salivation was the only abnormality in general condition observed in the male that died. All surviving males (11/12 males) exhibited transient salivation immediately after administration, and one male also exhibited perianal stool and loose stool.

2) Females:
- 0, 30, or 100 mg/kg group: no abnormalities in general condition were observed.
- 300 mg/kg group: immediately after administration transient salivation was observed from dosing day 11 onwards (12/12 females).
- 1000 mg/kg group: immediately after administration transient salivation was the only abnormality in general condition observed in the female that died. All surviving females exhibited transient salivation immediately after administration.

MORTALITY
1) Males:
- 0, 30, 100, or 300 mg/kg groups: no dead or dying males.
- 1000 mg/kg group: one male died on dosing day 27.

2) Females:
- 0, 30, 100 or 300 mg/kg group: no dead or dying females.
- 1000 mg/kg: one female died on day 19 of gestation.

BODY WEIGHT AND WEIGHT CHANGES
1) Males:
- 30, 100 and 300 mg/kg groups: body weights did not differ significantly from those in the control group on any measurement day.

2) Females:
- 30, 100, 300 and 1000 mg/kg mg/kg groups (before mating, mating period, and lactation period): body weights did not differ significantly from those in the control group on any measurement day.
- 30, 100 and 300 mg/kg groups (gestation period): body weights did not differ significantly from those in the control group on any measurement day.
- 1000 mg/kg group (gestation period): body weights measured on gestation day 21 tended to be lower than those in the control group (not statistically significant).

FOOD CONSUMPTION
1) Males:
- 30, 100 and 300 mg/kg groups: food consumption did not differ significantly from that in the control group on any measurement day.
- 1000 mg/kg group: food consumption on dosing day 3 was significantly lower than in the control group (p<0.05).

2) Females:
- 30, 100 and 300 mg/kg groups (before mating): food consumption did not differ significantly from that in the control group on any measurement day.
- 1000 mg/kg group (before mating): food consumption on dosing day 3 was significantly lower than in the control group (p<0.05).
- 30, 100, 300 and 1000 mg/kg mg/kg groups (gestation or lactation period): food consumption did not differ significantly from that in the control group on any measurement day.

URINALYSIS
1) Males:
- 30, 100 and 300 mg/kg groups: urine volume and specific gravity did not differ significantly from the control group.
- 30, 100, 300 and 1000 mg/kg mg/kg groups: urine colour, pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen and urinary sediment were more or less the same as in the control group.
- 1000 mg/kg group: urine volume was significantly higher and the urine specific gravity was significantly lower than in the control group (p<0.01).

2) Females:
- 30, 100, 300 and 1000 mg/kg mg/kg groups: urine volume or specific gravity did not differ significantly from the control group. The urine colour, pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen and urinary sediment were more or less the same as in the control group.

HAEMATOLOGY
1) Males:
- 30 and 100 mg/kg groups: no parameter measured differed significantly from that in the control group.
- 300 mg/kg group: MCH level was significantly higher than in the control group, but only slightly, and there was no difference in RBC (no toxicological effect).

2) Females:
- 100 and 300 mg/kg groups: no parameter measured differed significantly from that in the control group.
- 30 and 1000 mg/kg groups: MCV and MCH levels were significantly higher than in the control group (small differences compared to the control group; no difference in RBC; no toxicological effects).

CLINICAL BIOCHEMISTRY FINDINGS
1) Males:
- 300 mg/kg group:, no parameter measured differed significantly from that in the control group.
- 30 and 300 mg/kg groups: total bilirubin was significantly higher than in the control group (no significant difference was observed in the 300 or 1000 mg/kg group; not test item-related finding).

2) Females:
- 30, 100 and 300 mg/kg groups: ALP levels were significantly lower than in the control group (no significant difference was observed in the 1000 mg/kg group; not test item-related finding).

GROSS PATHOLOGICAL FINDINGS
1) Males:
- 0, 30 or 300 mg/kg groups: no abnormalities were observed.
- 1000 mg/kg group: 1/12 male exhibited a dark red spot on the glandular stomach mucosa, and 2/12 males exhibited ulceration of the glandular stomach mucosa. 1/12 male exhibited atrophy of the testis (right) and of the epididymis (right) (accidental findings). Adrenal hypertrophy was observed in the male that died.
- 100 mg/kg group: 1/12 male exhibited adhesion of the spleen (accidental finding).

2) Females
- 30, 100, 300 and 1000 mg/kg mg/kg groups: no abnormalities were observed.
- 1000 mg/kg group: pituitary tumour, atrophy of the thymus, dark red discolouration in the lung and adrenal hypertrophy were observed in the female that died.
ORGAN WEIGHTS
1) Males:
- 30 and 100 mg/kg groups: none of the relative or absolute organ weights differed significantly from those in the control group
- 300 mg/kg group: absolute testis weights were significantly higher than in the control group (no significant difference was observed in the 1000 mg/kg group; not test item-related finding).
- 1000 mg/kg group: absolute weights of the pituitary and heart were significantly lower, and the relative weights of the brain and testis were significantly higher, than in the control group (changes were attributed to difference in body weight compared to the control group; not test item-related findings).

2) Females:
- 30, 100 and 300 mg/kg groups: none of the relative or absolute organ weights differed significantly from those in the control group.
- 1000 mg/kg group: relative weights of the uterus were significantly higher than in the control group (changes were attributed to difference in body weight compared to the control group; not test item-related finding).

HISTOPATHOLOGICAL FINDINGS. NON-NEOPLASTIC
1) Males (surviving males):
1000 mg/kg group:
- thymus: very mild atrophy was observed in two males.
- stomach: moderate ulceration of the glandular stomach in one male, very mild erosion of the glandular stomach in one male, mild or moderate inflammatory cell infiltration of the glandular stomach submucosa in two males, mild haemorrhage of the glandular stomach submucosa in one male, and very mild vacuolisation of the forestomach epithelium in one male.
- liver: yellow-brown pigment deposition in the periportal hepatocytes (significant difference compared to control) in all six males, and yellow-brown pigment deposition in the periportal Kupffer cells in three males. The yellow-brown pigment deposition was very mild or mild. On special staining of the livers, bile staining afforded no staining, iron staining afforded mild or moderate periportal staining, and wear-and-tear pigment staining afforded very mild or mild periportal staining.

- kidney: very mild basophilic changes in the tubular epithelium in four males (significant difference compared to the control group).
- bone marrow: mild increased haematopoiesis in the femur was observed in one male.

The following changes were commonly observed in the control group, and did not differ in incidence between the control group and the administration groups, and so they were deemed accidental:
- eyeballs: dysplasia of the retina in one male.
- heart: focal histiocytic infiltration in one male.
- lungs: focal foam cell accumulation in two males.
- liver: focal hepatocyte necrosis in three males, bile duct proliferation in five males, and lymphoid cell infiltration in one male.
- bladder: subepithelial haemorrhage in one male.
- testes: atrophy of the seminiferous tubules in one male, Leydig's cell hyperplasia in one male, seminiferous tubule degeneration in one male, and exfoliated round spermatids in the seminiferous tubules in one male.
- epididymis: empty duct in one male.
- prostate: lymphoid cell infiltration in three males.

The following changes were commonly observed in the control group, and did not differ in incidence between the control group and the administration groups, and so they were deemed accidental:
- liver: bile duct proliferation in four males, and lymphoid cell infiltration in four males.

100 mg/kg group:
- spleen: extramedullary haematopoiesis in two males, and yellow-brown pigment deposition in the red pulp in all six males. The extramedullary haematopoiesis observed was very mild in the 100 mg/kg group. The yellow-brown pigment deposition in the red pulp was very mild or mild in the 100 mg/kg group.
-kidney: very mild basophilic changes in the tubular epithelium in one male.

The following changes were commonly observed in the control group, and did not differ in incidence between the control group and the administration groups, and so they were deemed accidental:
- liver: focal hepatocyte necrosis in three males, bile duct proliferation in one male, lymphoid cell infiltration in two males in the 100 mg/kg group, and yellow-brown pigment deposition in the centrilobular Kupffer cells in one male.
- spleen: fibrosis of the capsule due to adhesive inflammation in one male.

30 mg/kg group:
- spleen: extramedullary haematopoiesis in one male, and yellow-brown pigment deposition in the red pulp in all six males. The extramedullary haematopoiesis observed was very mild 30 mg/kg group. The yellow-brown pigment deposition in the red pulp was very mild in the 30 mg/kg group.
-kidney: very mild basophilic changes in the tubular epithelium in two males.

The following changes were commonly observed in the control group, and did not differ in incidence between the control group and the administration groups, and so they were deemed accidental:
- liver: focal hepatocyte necrosis in one male, bile duct proliferation in two males, and lymphoid cell infiltration in five males.

0 mg/kg group:
- spleen: extramedullary haematopoiesis in two males, and yellow-brown pigment deposition in the red pulp in all six males. The extramedullary haematopoiesis observed was very mild in the control group.
- heart: focal histiocytic infiltration in two males in the control group.
- liver: focal hepatocyte necrosis in one male, microgranuloma in two males, bile duct proliferation in three males, and lymphoid cell infiltration in four males
- pancreas: lobular atrophy in one male.
- bladder: subepithelial haemorrhage in one male.
- testes: seminiferous tubule degeneration in one male and exfoliated round spermatids in the seminiferous tubules in one male.
- prostate: lymphoid cell infiltration in three males.

No abnormalities were observed in the trachea, sublingual gland, submandibular gland, oesophagus, duodenum, coelenteron, ileum, caecum, colon, rectum, submandibular lymph nodes, mesenteric lymph nodes, seminal vesicles, pituitary, adrenals, thyroid, parathyroid, cerebrum, cerebellum, medulla, spinal cord, sciatic nerve, harderian gland or bones, in the control group or in the 1000 mg/kg group.

2) Males (dead)
1000 mg/kg group: one dead male had moderate postmortal changes in the tissue. The histological findings were as follows:
heart: mild mineral deposition.
lungs: mild congestion.
liver: very mild yellow-brown pigment deposition in periportal hepatocytes.
Due to the postmortal changes, no findings could be obtained for the adrenals that had exhibited abnormalities on necropsy.

3) Females (surviving females)
1000 mg/kg group:
- liver: all six females exhibited very mild yellow-brown pigment deposition in the periportal hepatocytes (significant difference compared to the control group). The yellow-brown pigment was such that on special staining of livers, bile staining afforded no staining, iron staining afforded mild or moderate periportal staining, and wear-and-tear pigment staining afforded very mild or mild periportal staining.
- spleen: extramedullary haematopoiesis and yellow-brown pigment deposition in the red pulp in all six females. The yellow-brown pigment deposition in the red pulp was moderate (severity differed from that in the control group). The extramedullary haematopoiesis was very mild to moderate.

300 mg/kg group:
- spleen: extramedullary haematopoiesis and yellow-brown pigment deposition in the red pulp in all six females. The yellow-brown pigment deposition in the red pulp was very mild or mild. The extramedullary haematopoiesis was very mild to moderate.

100 mg/kg group:
- spleen: extramedullary haematopoiesis and yellow-brown pigment deposition in the red pulp in all six females. The yellow-brown pigment deposition in the red pulp was very mild or mild. The extramedullary haematopoiesis was very mild to moderate.

30 mg/kg group:
- spleen: extramedullary haematopoiesis in six females, and yellow-brown pigment deposition in the red pulp in five females. The yellow-brown pigment deposition in the red pulp was very mild. The extramedullary haematopoiesis was mild or moderate.

0 mg/kg group:
- spleen: extramedullary haematopoiesis and yellow-brown pigment deposition in the red pulp in all six females. The yellow-brown pigment deposition in the red pulp was mild in the control group. The extramedullary haematopoiesis was very mild to moderate in the control.

The following changes were commonly observed in the control group, and did not differ in incidence between the control group and the administration groups, and so they were deemed accidental changes.
1000 mg/kg group:
- lungs: focal foam cell accumulation in one female.
- liver: microgranuloma in one female, lymphoid cell infiltration in two females, and bile duct proliferation in two females.
- kidneys: basophilic changes in the tubular epithelium in one female.
- bladder: ulceration in one female.

300 mg/kg group:
- liver: microgranuloma in one female, lymphoid cell infiltration in one female, and bile duct proliferation in three females
- kidneys: basophilic changes in the tubular epithelium in one female and lymphoid cell infiltration in one female.

100 mg/kg group:
- liver: Microgranuloma in one female in the 30 mg/kg group, one female in the 300 mg/kg group, and one female in the 1000 mg/kg group; lymphoid cell infiltration in three females in the control group, two females in the 30 mg/kg group, one female in the 300 mg/kg group, and two females in the 1000 mg/kg group; bile duct proliferation in three females in the control group, two females in the 30 mg/kg group, three females in the 100 mg/kg group, three females in the 300 mg/kg group and two females in the 1000 mg/kg group; periportal hepatocyte vacuolisation in one female in the control group.
- kidneys: lymphoid cell infiltration in one female.

30 mg/kg group:
- liver: microgranuloma in one female, lymphoid cell infiltration in two females, and bile duct proliferation in two females.
- kidneys: basophilic changes in the tubular epithelium in one female.

0 mg/kg group:
- heart: focal histiocytic infiltration in two females.
- lungs: focal foam cell accumulation in one female.
- liver: lymphoid cell infiltration in three females, bile duct proliferation in three females, and periportal hepatocyte vacuolisation in one female.
- kidneys: lymphoid cell infiltration in one female
- pituitary: anterior lobe cyst in one female.

No abnormalities were observed in the trachea, pancreas, sublingual gland, submandibular gland, oesophagus, stomach, duodenum, coelenteron, ileum, caecum, colon, rectum, thymus, submandibular lymph nodes, mesenteric lymph nodes, ovaries, uterus, vagina, adrenals, thyroid, parathyroid, cerebrum, cerebellum, medulla, spinal cord, sciatic nerve, harderian gland, bones, bone marrow or mammary glands, in the control group or in the 1000 mg/kg group.

4) Females (dead)
1000 mg/kg group: one dead female had severe postmortal changes in the tissue. The histological findings were as follows.
- lungs: mild congestion and mild oedema.
- liver: very mild mineral deposition.
Due to the postmortal changes, no findings could be obtained for the pituitary, thymus or adrenals that had exhibited abnormalities on necropsy.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effect

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

iron

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

other: value for element iron, which was recalculated from the value obtained for the test material

Critical effects observed:

not specified

Conclusions:

In the current combined repeated dose toxicity study with the reproduction developmental toxicity screening test, groups of 12 male and 12 female Sprague-Dawley rats were administered iron sulfate heptahydrate via gavage at dose levels of 30, 100, 300 and 1000 mg/kg bw/day (equivalent to 6, 20, 60 and 201 mg Fe/kg bw/day). Males and females were treated with the substance for a duration of 49 days (14 days before mating and 35 days after mating) and 42 - 47 days (14 days before mating, throughout the mating period and the gestation period until lactation day 5), respectively. A vehicle control group was run concurrently.

After oral administration of 30, 100, 300 and 1000 mg/kg/day of the test item no effects were observed on food consumption and gross pathology.

General observation revealed salivation in males and females in the ≥300 mg/kg groups. This was transient and only observed immediately after administration, and there were no neurological symptoms such as convulsion or morphological changes to the salivary glands, and so the salivation was attributed to irritation by the test substance, and was not deemed to be a symptom of toxicity.

After the administration 1000 mg/kg/day of the test item, one male and one female died. These animals had exhibited salivation on observation of general condition. Necropsy of the dead animals revealed adrenal hypertrophy in the male and pituitary tumour, atrophy of the thymus, dark red discolouration of the lungs and adrenal hypertrophy in the female. Histological examination revealed mineral deposition in the heart, congestion of the lungs and yellow-brown pigment deposition in the periportal hepatocytes in the male and congestion and oedema in the lungs and mineral deposition in the liver in the female.

In addition to the findings described above for the 1000 mg/kg/day dose group, body weights in the 1000 mg/kg group were somewhat low throughout the administration period in the males, and tended to be low in the late gestation period in the females. Furthermore, temporarily low food consumption was observed in males and females in the this group. Urine tests revealed high urine volume and low specific gravity in males of the1000 mg/kg/day group, but no changes attributable to test item administration were observed in the females. Haematology tests revealed low RBC and APTT values, and high MCV, MCH and reticulocyte levels in males, but no changes attributable to administration were observed in the females. Blood biochemistry test revealed low total protein, albumin and Ca levels, and high ALT, γ-GTP and A/G levels in males and high γ-GTP and organic phosphorus levels in females. The necropsies revealed dark red spots and ulceration of the glandular stomach mucosa in males in the 1000 mg/kg group, but no changes caused by administration were observed in the females. Further, organ weight measurements revealed high absolute and relative adrenal weights and high relative liver weights in males in the 1000 mg/kg group, and high absolute and relative liver weights in females in the 1000 mg/kg group. Lastly, the histological investigation of the 1000 mg/kg/day group revelaed that the thymus findings were atrophy of the thymus in two males. The stomach findings were ulceration of the glandular stomach in one male, erosion of the glandular stomach in one male, inflammatory cell infiltration of the glandular stomach submucosa in two males, haemorrhage of the glandular stomach submucosa in one male, and vacuolisation of the forestomach epithelium in one male. The liver findings were yellow-brown pigment deposition in periportal hepatocytes in all six males, and yellow-brown pigment deposition in periportal Kupffer cells in three males and yellow-brown pigment deposition in periportal hepatocytes in all six females. The spleen findings were extramedullary haematopoiesis in four males, and yellow-brown pigment deposition in the red pulp in all six males and yellow-brown pigment deposition in the red pulp in all six females. These findings were observed at greater severity in the high dose group than in the control group. The kidney findings were basophilic changes in the tubular epithelium in four males. The bone marrow findings were increased haematopoiesis in the femur in one male.

After the administration 300 mg/kg/day of the test item, blood biochemistry tests revealed high organic phosphorus levels in females in the 300 mg/kg group. Furthermore, the histopathological investigation revealed increased extramedullary haematopoiesis in the spleen in five males.

In conclusion, a No Observed Adverse Effect Level (NOAEL) for systemic toxicity of 300 mg/kg/day (equivalent to 60 mg Fe/kg bw/day) was concluded for both sexes based on the increased relative liver weight and increased gamma glutamylpeptidase in males and females at the 1000 mg/kg/day dose level.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

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

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. However, the stated food consumption finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group (test week 11): 51.16 ± 3.08 g/kg bw/day
Treatment group (test week 11): 48.27 ± 2.32 g/kg bw/day

- 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.
Control group (test week 9, males): 52.48 ± 3.84 g/kg bw/day
Treatment group (test week 9, males): 49.61 ± 2.38 g/kg bw/day
Control group (test week 11, males): 51.16 ± 3.08 g/kg bw/day
Treatment group (test week 11, males): 48.79 ± 2.43 g/kg bw/day

Control group (test week 14, females): 66.87 ± 4.31 g/kg bw/day
Treatment group (test week 14, females): 60.47 ± 3.73 g/kg bw/day
Control group (test week 15, females): 66.17 ± 4.82 g/kg bw/day
Treatment group (test week 15, females): 57.65 ± 4.94 g/kg bw/day
Control group (test week 16, females): 65.22 ± 3.11 g/kg bw/day
Treatment group (test week 16, females): 57.28 ± 5.67 g/kg bw/day

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. However, the stated food consumption findings are within in the normal range, typical for that strain and the age of the animals (except for test weeks 1 and 4; see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.

Control group (test week 1, males): 61.90 ± 2.28 g/kg bw/day
Treatment group (test week 1, males): 66.70 ± 3.49 g/kg bw/day
Control group (test week 2, males): 60.63 ± 2.46 g/kg bw/day
Treatment group (test week 2, males): 64.46 ± 4.21 g/kg bw/day
Control group (test week 4, males): 55.01 ± 2.09 g/kg bw/day
Treatment group (test week 4, males): 58.77 ± 2.83 g/kg bw/day
Control group (test week 5, males): 55.60 ± 1.30 g/kg bw/day
Treatment group (test week 5, males): 59.39 ± 2.94 g/kg bw/day
Control group (test week 6, males): 54.18 ± 2.17 g/kg bw/day
Treatment group (test week 6, males): 59.16 ± 2.53 g/kg bw/day
Control group (test week 7, males): 49.21 ± 2.86 g/kg bw/day
Treatment group (test week 7, males): 54.48 ± 2.65 g/kg bw/day
Control group (test week 12, males): 47.65 ± 1.27 g/kg bw/day
Treatment group (test week 12, males): 50.78 ± 2.42 g/kg bw/day

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. However, the stated haematological finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance and lacking dose-dependency.
Control group (test day 91; x10E3/µL): 0.154 ± 0.078
Treatment group (test day 91; x10E3/µL): 0.092 ± 0.035

- 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. However, the stated haematological finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group (test day 91; x10E3/µL): 0.154 ± 0.078
Treatment group (test day 91; x10E3/µL): 0.086 ± 0.059

- 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. However, the stated haematological finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group (test day 119): 15.90 ± 0.95 seconds
Treatment group (test day 119): 14.82 ± 0.41 seconds

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. However, the stated clinical chemistry finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Control group (test day 91): 2.86 ± 0.48 µmol/L
Treatment group (test day 91): 3.58 ± 0.48 µmol/L

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.
Control group (test day 91): 2.3502 ± 0.2364 ng/mL
Treatment group (test day 91): 2.0813 ± 0.2242 ng/mL

- 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.
Control group (test day 91): 1.0934 ± 0.5241 ng/mL
Treatment group (test day 91): 0.1725 ± 0.1574 ng/mL

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. However, the stated urinalysis finding is within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below).The slight alteration in comparison to control animals is without any biological relevance.
Control group (test day 91): 27.50 ± 8.53 mL/kg bw/24 h
Treatment group (test day 91): 19.82 ± 11.85 mL/kg bw/24 h

- 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 ≤ 0.05) decrease in relative volume was observed for female rats on test days 91 and 119. However, the stated urinalysis findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alterations in comparison to control animals are without any biological relevance.

Specific gravity
Control group (test day 119): 1.0454 ± 0.0195 g/mL
Treatment group (test day 119): 1.0736 ± 0.0158 g/mL

Relative volume
Control group (test day 91): 27.50 ± 8.53 mL/kg bw/24 h
Treatment group (test day 91): 15.96 ± 7.13 mL/kg bw/24 h
Control group (test day 119): 25.55 ± 11.08 mL/kg bw/24 h
Treatment group (test day 119): 11.51 ± 3.40 mL/kg bw/24 h

- 1000 mg/kg bw/day: a statistically significant (p ≤ 0.01 or p ≤ 0.05) decrease in pH was observed for female rats on test day 119. The slight alterations in comparison to control animals are without any biological relevance.
Control group (test day 119): 6.40 ± 0.27
Treatment group (test day 119): 5.90 ± 0.19

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.
Control group (test week 13): 223.4 ± 61.4 g
Treatment group (test week 13): 121.1 ± 21.8 g

- 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.
Control group (test week 13): 223.4 ± 61.4 g
Treatment group (test week 13): 155.5 ± 37.6 g

- 1000 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.
Control group (test week 13): 223.4 ± 61.4 g
Treatment group (test week 13): 295.6 ± 27.3 g

- 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.
Hind leg splay
Control group (test week 13): 6.8 ± 0.8 cm
Treatment group (test week 13): 6.1 ± 0.6 cm

Hindlimb grip strength
Control group (test week 13): 128.8 ± 64.9 g
Treatment group (test week 13): 76.3 ± 19.5 g

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.
Control group (test week 17): 42.5 ± 10.7 g
Treatment group (test week 17): 70.5 ± 17.0 g

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. However, the stated absolute organ weight findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Left testis weight
Control group (test 119): 1.708 ± 0.145 g
Treatment group (test day 119): 1.964 ± 0.099 g

Left ovarian weight
Control group (test 119): 0.0380 ± 0.0063 g
Treatment group (test day 119): 0.0548 ± 0.0107 g

Right ovarian weight
Control group (test 119): 0.0468 ± 0.0094 g
Treatment group (test day 119): 0.0634 ± 0.0079 g

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. However, the stated relative organ weight findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). The slight alteration in comparison to control animals is without any biological relevance.
Pituitary weight
Control group (test 91): 0.0635 ± 0.0146 g
Treatment group (test day 91): 0.0781 ± 0.0184 g

Left ovarian weight
Control group (test 119): 0.1377 ± 0.0227 g
Treatment group (test day 119): 0.1894 ± 0.228 g

Right ovarian weight
Control group (test 119): 0.1708 ± 0.0331g
Treatment group (test day 119): 0.2206 ± 0.0194 g

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.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

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.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Experimental set up and methods were not described in detail. Test material was insufficiently characterised (no purity, impurities). Actual concentration of iron in the diet was not analysed. A rationale for the selection of the dose levels was not given. Study duration was too short for a sub-chronic RDT study. Only males were tested and the no. of animals per group was too low for a statistical analysis. Male rats were too young. Clinical signs or cage-side observations were not performed. Weekly body weights during study were missing. Food consumption was not recorded. An ophthalmological examination or heamatology were not performed. Clinical biochemistry and histopathological examination were incomplete. Information about test animals (no age or acclimation period) and environmental conditions (no photoperiod, temperature or humidity) were insufficient. A complete necropsy was performed but the results were not presented. Results of histopathological examination were not shown in tabular form.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male Sprague–Dawley rats were fed either Fe-deficient or carbonyl Fe supplemented diets with 35 (control), 350, 3500 and 20000mg Fe/g for 12 weeks to examine the effects of increasing levels of Fe on serum fatty acids, cholesterol, triacylglycerol, liver and heart. At the end of study, animals were weighed, a complete necropsy was performed, the liver and heart were weighed and tissue specimens were histopathologically examined. Furthermore, the non-haem Fe was determined in liver and heart tissue, as well as the concentration of iron and total Fe-binding capacity (TIBC) in serum. In addition, the concentration of the liver conjugated diene was analysed. Also in serum, the levels of cholesterol and triacylglycerol were examined, as well as the levels of phospholipid fatty acids and free fatty acids saturated, unsaturated and in total, and the ratio of saturated/unsaturated phospholipid fatty acids or free fatty acids.

GLP compliance:

not specified

Remarks:

in the publication.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ISP Technologies, Inc., Wayne, NJ, USA

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Inc., Altamont, NY, USA
- Age at study initiation: weanling rats
- Weight at study initiation: approx. 42 g
- Housing: individually housed in stainless steel cages
- Diet (ad libitum): AIN-76A diets (supplier: Dyets, Inc., Bethlehem, PA, USA), diet (without added Fe) contained (g/kg) casein 200.0, DL-methionine 3.0, cornstarch 150.0, glucose 500.0, fibre Celufil 50.0, AIN-76 mineral mix (without Fe) 35.0 and AIN-76A vitamin mix 10.0 with 50.0mg menadione/kg and 2.0 g choline bitartrate/kg.
- Water (ad libitum): distilled, deionized water

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

daily

Dose / conc.:

4 other: µg Fe/g diet

Remarks:

Fe-deficient group

Dose / conc.:

35 other: µg Fe/g diet

Remarks:

Control group

Dose / conc.:

350 other: µg Fe/g diet

Dose / conc.:

3 500 other: µg Fe/g diet

Dose / conc.:

20 000 other: µg Fe/g diet

No. of animals per sex per dose:

8 - 10 male rats

Control animals:

yes, concurrent no treatment

yes, plain diet

Positive control:

Not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified
DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: the initial and final body weight were deterimed.
- How many animals: all rats in the Fe-deficient, control and treatment groups

FOOD CONSUMPTION AND COMPOUND INTAKE: Not specified
WATER CONSUMPTION AND COMPOUND INTAKE: Not specified
HAEMATOLOGY: Not specified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 12 week exposure
- Animals fasted: not specified
- How many animals: 9 rats per treatment group except for the groups receiving 35 and 350 mg Fe/g diet (n=8) and 3500 mg Fe/g diet (n=7).
- Parameters examined: in serum the levels of cholesterol, triacylglycerol, phospholipid fatty acids and free fatty acids saturated, unsaturated and in total, and the ratio of saturated/unsaturated phospholipid fatty acids or free fatty acids.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the study, the rats were anaesthetized by intramuscular injection of 5mg ketamine hydrochloride per 100 g body weight, and decapitated. A complete necropsy was performed on each animal.

ORGAN WEIGHTS: Yes
Organ weights of liver and heart were determined.

HISTOPATHOLOGY: Yes
At the end of study, tissues were fixed in 10% neutral buffered formalin. Tissue specimens were then trimmed, processed through ascending ethanol series and toluene, embedded in paraffin and sectioned at 7mm. One set of tissues was stained with haematoxylin and eosin (H&E) and one set with Perl’s Prussian blue for Fe, which involves colourless potassium ferrocyanide reacting with ferric ions to create an insoluble blue ferric ferrocyanide (Thompson, S.W. et al. (1966); Luna, L.G. (1968)).*

*References:
- Thompson SW & Hunt RD (1966): Selected Histochemical and Histopathological Methods. Springfield, IL: Charles C. Thomas.
- Luna LG (1968) Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. New York: McGraw-Hill.

Other examinations:

NON-HEME IRON:
non-haem Fe content was determined by the method of Whittaker et al. (1996a).*

*Reference:
- Whittaker P, Hines FA, Robl MG & Dunkel VC (1996a): Histopathological evaluation of liver, pancreas, spleen, and heart from iron-overloaded Sprague–Dawley rats. Toxicologic Pathology 24, 558–563.

Statistics:

Differences among dietary treatment groups were assessed by one-way ANOVA, using the ABstat (1993) general linear model program. The Scheffe´ multiple comparison method was used to differentiate among means for variables that were significantly affected by the treatments (Snedecor & Cochran, 1980). Values are expressed as mean with their standard errors. Correlation coefficients were determined using Pearson’s product moment correlation matrix (Snedecor & Cochran, 1980).

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

- 3500 and 20000 µg Fe/g: 2 animals on 3500 µg/g and 5 on highest dose, died before termination of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 3500 and 20000 µg/g: final body weights decreased statistically significant compared to controls (p<0.001).

Please refer to the attached background material (table 2) in section overall "Remarks, attachments".

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 3500 and 20000 µg/g: serum cholesterol levels were statistically significant increased in comparison to controls (p<0.01).

Please refer to the attached background material (table 3) in section overall "Remarks, attachments".

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 3500 and 20000 µg/g: relative heart and liver weights were statistically significant increased in comparison to control group (p<0.001).

Please refer to the attached background material (table 2) in section overall "Remarks, attachments".

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 3500 and 20000 µg/g: the incidence of severity of cardiomyopathy increased with higher dietary concentrations of Fe and was characterized by a spectrum of lesions. These included small foci of degenerated myofibres with variable numbers of mononuclear and polymorphonuclear inflammatory cells and a few fibroblasts with or without oedema, to extensive areas of degeneration or necrosis of myofibres. There were areas of fibrosis near these degenerative or necrotic lesions, as well as disruption of the ventricular or atrial walls, and thrombi formation. 5 of the 7 animals which died before study termination had heart damage, which included Fe in the cytoplasm of the myocardial fibres, haemorrhagic necrosis, epicardial damage and clot formation.

- 20000 µg/g: hepatocellular hypertrophy, manifested as enlargement of the individual hepatocytes, was observed in 6/10 animals. The enlarged hepatocytes, located predominantly in the periportal region of the liver lobules, were characterized by an increased amount of cytoplasm in relation to the nucleus and a large amount of granular cytoplasmic Fe.

- Control group: a small amount of Fe was observed only in macrophages found in the liver sinuses.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

NON-HAEM IRON:
- 3500 and 20000 µg/g: non-haem Fe levels in liver were statistically significant increased in the 3500 and 20000 µg/g group and in the heart of rats from the 20000 µg/g group compared to controls (p<0.001).

Please refer to the attached background material (table 2) in section overall "Remarks, attachments".

Details on results:

BODY WEIGHT AND WEIGHT CHANGES:
- 350, 3500 20000 µg/g: final body weight decreased dose-related in all treated group.

CLINICAL BIOCHEMISTRY:
- 350, 3500 and 20000 µg/g: serum cholesterol level increased dose-related in all treated groups. The serum Fe and TIBC of Fe-overloaded rats were not significantly different from those of control animals.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN WEIGHT / BODY WEIGHT RATIOS:
- 350, 3500 and 20000 µg/g: relative liver weights increased dose-related in all treated groups.

IORN CONTENT IN SERUM:
- 350, 3500 and 20000 µg/g: iron content serum decreased dose-related in all treatment groups.

NON HEAM IRON:
- 350, 3500 and 20000 µg/g: non-haem Fe in heart and liver increased dose-related in all treated groups.

Dose descriptor:

NOAEL

Effect level:

> 350 other: µg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

Dose descriptor:

NOAEL

Effect level:

> 17.5 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

Remarks on result:

other: Calculated based on the amount of Fe incorporated in the feed, the final body weight and the calculated feed intake (approximately 5 % of body weight, based on the Guidance on the Application of the CLP (version 5)).

Critical effects observed:

not specified

Conclusions:

NOAEL (male rat): > 350 μg Fe/g diet (equivalent to 17.5 mg Fe/kg bw)

Whittaker, P. et al. (2001) fed male Sprague–Dawley rats either with Fe-deficient or carbonyl Fe supplemented diets with 35 (control), 350, 3500 and 20000 µg Fe/g for 12 weeks to examine the effects of increasing levels of Fe on serum fatty acids, cholesterol, triacylglycerol, liver and heart. At the end of study, animals were weighed, a complete necropsy was performed, the liver and heart were weighed and tissue specimens were histopathologically examined. Furthermore, the non-haem Fe was determined in liver and heart tissue, as well as the concentration of iron and total Fe-binding capacity (TIBC) in serum. In addition, the concentration of the liver conjugated diene was analysed. Also in serum, the levels of cholesterol and triacylglycerol were examined, as well as the levels of phospholipid fatty acids and free fatty acids saturated, unsaturated and in total, and the ratio of saturated/unsaturated phospholipid fatty acids or free fatty acids.

Before termination of the study, 2 animals on 3500 µg/g and 5 on 20000 µg/g dose died. In these both dose groups, the final body weights decreased compared to controls (35 µg/g). The serum cholesterol levels and non-haem Fe levels in liver were increased in the 3500 and 20000 µg/g groups, as well as the non-haem Fe in heart of rats from the 20000 µg/g group. Furthermore, the relative heart and liver weights were increased in the 3500 and 20000 µg/g groups in compared to controls. There were lesions in the heart of animals from these both dose groups. The incidence of severity of cardiomyopathy increased with higher dietary concentrations of Fe and was characterized by a spectrum of lesions. Five of the seven animals which died before study termination had heart damage, which included Fe in the cytoplasm of the myocardial fibres, haemorrhagic necrosis, epicardial damage and clot formation. In addition, in the liver of animals (6/10 rats) from the 20000 µg/g group hepatocellular hypertrophy was observed, manifested as enlargement of the individual hepatocytes.
Deficiencies:
The experimental set up and methods were not described in detail. Test material was insufficiently characterised (no purity, impurities). The actual concentration of iron in the diet was not analysed. A rationale for the selection of the dose levels was not given. The study duration (12 weeks) was too short for a sub-chronic RDT study. Only males were tested and the no. of animals per group (n=8-10) was too low for a statistical analysis. Based on the initial body weight of the males of approx. 42 g, an age of approx. 3 weeks is assumed before the start of the study (according to the growth chart of Sprague-Dawley rats). However, according to OECD 408 (1998), dosing should begin as soon as possible after weaning and the animals should be young healthy adults. If the animals were around 3 weeks old at the beginning of the study, that would be too young. At 3 weeks of age, they had not yet completed the weaning phase, which lasts 4 weeks for rats. Furthermore, animals were not observed for clinical signs, and cage-side observations were not performed. In addition, only the initial and final body weights were determined but the animals were not weighed weekly during the study. Food consumption was not recorded. An ophthalmological examination or heamatology were not performed. During the clinical biochemistry, the levels of cholesterol and other lipids were analysed in serum, but the sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, creatinine, total protein and albumin, and more than two enzymes indicative of hepatocellular effects were not investigated. The histopathological examination was incomplete since only the liver and heart were examined. Information about test animals (no age or acclimation period) and environmental conditions (no photoperiod, temperature or humidity) were insufficient. A complete necropsy was performed but the results were not presented in the publication. The results of the histopathological examination were not shown in tabular form.

Reference 6

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low. Rats were not observed for clinical signs, and cage-side observations were not performed. Rats were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, haematology or clinical biochemistry were not performed. Histopathological examination was incomplete. Information about test animals and environmental conditions were insufficient. Organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

F344 rats were fed to carbonyl iron at concentrations of 1500, 3500, 5000 and 10000 µg Fe/g in diet for 90 days. At the end of study, food consumption and final body weight were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined (liver, pancreas, spleen, heart, kidney and testis epithelium). Furthermore, the concentrations of non-heme iron were determined. The cell proliferation in liver and cells in Langerhans islets were evaluated.

GLP compliance:

not specified

Remarks:

in publication.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ISP Technologies, Inc. Wayne, NJ

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks old
- Housing: animals were housed in polycarbonate cages, 2 rats/cage
- Diet (ad libitum): AIN-76A diet (source: Dyets, Inc., Bethlehem, PA), which contained (g/kg) casein 200.0, DL-methionine 3.0, cornstarch 150.00, glucose 500.0, fiber Celufil 50.0, AIN-76 mineral mix 35.0, and AIN-76A vitamin mix 10.0 with 50.0 mg menadione/kg and 2.0 g choline bitartrate/kg.
- Water (ad libitum): distilled, deionized water

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
Carbonyl Fe was incorporated into the AIN-76A diet.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

35 other: µg Fe/g diet

Remarks:

Control

Dose / conc.:

1 500 other: µg Fe/g diet

Dose / conc.:

3 500 other: µg Fe/g diet

Dose / conc.:

5 000 other: µg Fe/g diet

Dose / conc.:

10 000 other: µg Fe/g diet

No. of animals per sex per dose:

12 males

Control animals:

yes, plain diet

Details on study design:

- Section schedule rationale (if not random): animals were initially divided into 5 groups of approximately equal mean body weight.

Positive control:

Not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule (mortality): not specified

DETAILED CLINICAL OBSERVATIONS: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not specified

FOOD EFFICIENCY: not specified
WATER CONSUMPTION: not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
PLASMA/SERUM HORMONES/LIPIDS: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

Sacrifice and pathology:

GROSS PATHOLOGY: yes
A complete necropsy was performed on each animal.

ORGAN WEIGHT: yes
Different organs (liver, heart, kidneys, thymus, brain) were weighed. Liver was weighed for determination of absolute and relative liver weight.

HISTOPATHOLOGY: yes
Tissues were fixed in 10 % neutral buffered formalin. Tissue specimens were then trimmed, processed through an ascending ethanol series and xylene, embedded in paraffin, and sectioned at 5 µm. One set of tissues was stained with haematoxylin and eosin (H&E). Another set of the pancreas, liver, spleen, kidney, and heart was reacted with Perl’s Prussian blue for Fe, which involves colourless potassium ferrocyanide reacting with ferric ions to create an insoluble blue ferric ferrocyanide (Luna, L.G. et al. (1968), Thompson, S.W. et al. (1966)).*
Pancreas Morphometry: to ensure uniform diameter of sections of equivalent area, pancreatic tissues were adhered to filter paper and fixed in 10 % neutral buffered formalin. Procedures for embedding, sectioning, staining, and evaluating the pancreas were standardized to assure uniformity. Five-micron sections were mounted on positively charged slides and immunostained by using the StreptAvidin peroxidase detection system with DAB (3,3’-diaminobenzidine) as the chromogen substrate and Mayer’s haematoxylin as the counterstain. Sections were incubated with a polyclonal antibody to insulin (1:600 dilution) (Dako Corp.). Cells were counted and areas measured by using the Optimas Image Analysis System. All islets present in a representative section of each pancreas were evaluated for β-cell number and islet area.

*References:
- Luna LG (1968): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill, New York.
- Thompson SW and Hunt RD (1966): Selected Histochemical and Histopathological Methods. Charles C Thomas, Springfield, Illinois.

Other examinations:

NON-HEME IRON:
- Time schedule for examinations: at study termination (after 90 days of exposure).
- Procedure: non-heme Fe was determined in livers by the bathophenanthroline reaction and expressed as [µg Fe/g liver]. Approx. 1 g of each previously blotted liver was weighed and placed in a 50-ml polypropylene centrifuge tube, and distilled, deionized water was added to bring the volume to 15 ml. The tissue was then homogenized for 30 sec with a Polytron. A 3-ml sample of the homogenate was transferred to another 50-ml centrifuge tube, and 10 ml of acid reagent was added (6 M HCI and 1.2 M trichloroacetic acid, 1:1, v/v) and mixed well. This mixture was then heated in an oven at 65 °C for 20 hr, cooled, and centrifuged at 1,500xg for 20 min. Duplicate 0.2-ml aliquots of the supernatant fraction were pipetted into small polypropylene tubes, and 1.8 ml of freshly prepared colour reagent was added, mixed, and incubated for 10 min at room temperature. Absorbance was determined spectrophotometrically at 535 nm, and Fe concentration (µg Fe/ml) was determined by reference to a standard curve. The bathophenanthroline colour reagent (which was protected from light) was prepared by dissolving 62.5 mg bathophenanthroline disulfonic acid and 0.25 ml thioglycolic acid in distilled, deionized water and diluting to 25 ml. The final colour reagent was a solution of the bathophenanthroline colour reagent, saturated sodium acetate (4.5 M), and distilled, deionized water (1:20:20 by volume).

Statistics:

Differences among dietary treatment groups were assessed by 1-way analysis of variance (ANOVA), using the ABstat general linear model program. The Duncan multiple comparison method was used to differentiate among means for variables that were significantly affected by the treatments. Values are expressed as meant- SEM. Correlation coefficients were determined by using Pearson’s product moment correlation matrix. Mean organ: brain weight ratios were compared by 2-sided Student’s t-tests. Immunohistochemical assays and pancreas morphometry data were compared by ANOVA with the Holm’s correction.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

- 10000 µg Fe/g: 9 of 12 rats died before end of the 90-day treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1500, 3500, 5000 and 10000 µg Fe/g: body weight decreased statistically significant in a dose-dependent manner among the rats and the weight gain was reduced in rats compared to the controls (p<0.001).
Please refer to the attached background material (table V) in section overall "Remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 1500, 3500, 5000 and 10000 µg/g: food consumption in rats was statistically significantly reduced (p<0.001) compared to control group.
Please refer to the attached background material (table V) in section overall "Remarks, attachments".

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 1500, 3500, 5000 and 10000 µg Fe/g: absolute liver weights were significantly decreased in rats at the 10000 µg Fe/g dose level (p<0.001), only. The relative liver weights increased dose-related and were statistically significant different in all dose groups compared to controls (p<0.001).
- Heart weights were increased.
- Thymic weights in sacrificed rats were very low.
- 10000 µg Fe/g: heart:brain weight ratios were increased in rats.

Please refer to the attached background material in section overall "Remarks, attachments".

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
- 5000 and 10000 µg Fe/g: presence of iron and hemosiderin in liver primarily in hepatocytes in periportal areas and also in some Kupffer cells and some periportal macrophages (siderosis, average severity score 2.1 – 3.6).
- 10000 µg Fe/g: all rats showed minimal to moderate periportal hepatocyte hypertrophy (score 2.3).

Pancreas:
- 1500, 3500, 5000 and 10000 µg Fe/g: minimal Fe accumulation in acinar cells. Moderate to severe parenchymal degeneration in the pancreas (loss of acinar cells and islets, fat infiltration) in rats fed with 10000 µg Fe/g. Similar but less severe effects observed even with the lowest dose level.
- Pancreatic morphometry: islet area and number were quantified in rats fed with the 3500 µg Fe/g containing diet, because of a distorted structure of the islets at 5000 µg Fe/g. Please refer to the attached background material (table V) in section "Overall remarks, attachments".

Spleen:
- 5000 and 10000 µg Fe/g: lymphocyte depletion observed in rats (average severity score: 4.0 for 10000 µg Fe/g and 2.0 for 5000 µg Fe/g).

Kidney:
- control and 1500, 3500, 5000 and 10000 µg Fe/g: severity of kidney degeneration in control animals was minimal, but all rats in the group were affected (score of 1.0). In contrast, there was marked nephropathy in rats fed 10000 µg Fe/g, all of which were affected (score of 4.0). Early death in 9/12 rats fed 10000 µg Fe/g was attributed to this severe nephropathy. All rats fed 5000 µg Fe/g also had an increased severity of nephropathy (score of 1.9).
- 10000 µg Fe/g: 3 rats that survived until sacrifice revealed Fe granules in glomerular and tubular epithelium. The extent of Fe deposition varied among animals (score 2.0).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LIVER NON-HEME IRON:
- 1500, 3500, 5000 and 10000 µg Fe/g: dose-related and statistically significant increase in liver non-heme iron was observed (exposed groups: 685 - 1848 µg/g; control: 262 ± 37 µg/g) compared to controls.
Please refer to the attached background material (table V) in section overall "Remarks, attachments".

Details on results:

HISTOPATHOLOGICAL FINDINGS:
- Liver: similar hypertrophic changes in liver were not observed in control rats or in rats fed the 5,000 µg Fe/g diet.
- Heart: cardiomyopathy occurred more frequently in control animals (10/12 rats, with an average severity grade of 1.1) than in rats fed 5000 µg Fe/g (3/12 rats, average severity grade of 1.0). Of the 12 hearts examined from rats fed 10000 µg Fe/g, cardiomyopathy (severity grade 1.0) was observed in only 1 animal. Any increase in myocyte volume or number was not detectable.

Dose descriptor:

LOAEL

Effect level:

68.07 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: The effect level [mg/kg bw] was calculated based on the final body weight and feed intake. See the calculations in the field "Any other information on materials and methods incl. tables".

Dose descriptor:

LOAEL

Effect level:

1 500 other: µg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

LOAEL (male rat): 1500 µg Fe/g diet (equivalent to 68.07 mg Fe/kg bw)

Whittaker, P. et al. investigated the effects of carbonyl iron in F344 rats fed at concentrations of 1500, 3500, 5000 and 10000 µg Fe/g in diet for 90 days. At the end of study, food consumption and final body weight were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined. Furthermore, the concentrations of non-heme iron were determined.
According to the authors, 9 of 12 rats died in the 10000 µg Fe/g dose group before end of the 90-day treatment period. The body weights of rats in the 3500 - 10000 µg Fe/g group were reduced. There was a dose-related increase in liver non-heme Fe form the 3500 µg Fe/g group, and the Fe was stored in hepatocytes predominantly in the periportal region. Relative liver weights were dose-related and significantly increased in all dose groups. A significant hypertrophy of the hepatocytes was observed in rats fed 10000 µg Fe/g diet. There was also pancreatic atrophy with loss of both endocrine and exocrine tissue. In addition, rats in the 10000 µg Fe/g dose group had markedly exacerbated dose-dependent nephropathy and changes in glomerular and tubular epithelium associated with Fe accumulation. The rats also showed degeneration of the germinal epithelium of the testis, formation of multinucleated giant cells, and lack of mature sperm.


Deficiencies:
The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low for a statistical analysis. Animals were not observed for clinical signs, and cage-side observations were not performed. In addition, animals were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, heamatology or clinical biochemistry were not performed. The histopathological examination was incomplete since trachea, lungs, aorta, gonads, oesaphagus, stomach, duodenum, jejunm, ileum, caecum, colon, rectum, urinary bladder, peripheral nerve, sternum with bone marrow were not examined. Information about test animals (source, acclimation period, initial body weight) and environmental conditions (photoperiod, temperature, humidity) were insufficient. The results of the histopathological examination were not shown in tabular form, and the organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low. Animals were not observed for clinical signs, and cage-side observations were not performed. Mice were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, haematology or clinical biochemistry were not performed. Histopathological examination was incomplete. Information about test animals and environmental conditions were insufficient. Organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

B6C3F1 mice were fed to carbonyl iron at concentrations of 1500, 3500, 5000 and 10000 μg Fe/g in diet for 90 days. At the end of study, food consumption and final body weights were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined (liver, pancreas, spleen, heart, kidney and testis epithelium). Furthermore, the concentrations of non-heme iron were determined. The cell proliferation in liver and cells in Langerhans islets were evaluated.

GLP compliance:

not specified

Remarks:

in publication.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ISP Technologies, Inc. Wayne, NJ

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks old
- Housing: animals were housed in polycarbonate cages, 2 mice/cage
- Diet (ad libitum): AIN-76A diet (source: Dyets, Inc., Bethlehem, PA), which contained (g/kg) casein 200.0, DL-methionine 3.0, cornstarch 150.00, glucose 500.0, fiber Celufil 50.0, AIN-76 mineral mix 35.0, and AIN-76A vitamin mix 10.0 with 50.0 mg menadione/kg and 2.0 g choline bitartrate/kg.
- Water (ad libitum): distilled, deionized water

Route of administration:

oral: feed

Details on route of administration:

Not specified

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

35 other: μg Fe/g diet

Remarks:

Control

Dose / conc.:

1 500 other: μg Fe/g diet

Dose / conc.:

3 500 other: μg Fe/g diet

Dose / conc.:

5 000 other: μg Fe/g diet

Dose / conc.:

10 000 other: μg Fe/g diet

No. of animals per sex per dose:

12 males

Control animals:

yes, plain diet

Details on study design:

- Section schedule rationale (if not random): animals were initially divided into 5 groups of approximately equal mean body weight.

Positive control:

Not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule (mortality): not specified
DETAILED CLINICAL OBSERVATIONS: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not specified

FOOD EFFICIENCY: not specified
WATER CONSUMPTION: not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
PLASMA/SERUM HORMONES/LIPIDS: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

Sacrifice and pathology:

GROSS PATHOLOGY: yes
A complete necropsy was performed on each animal.

ORGAN WEIGHT: yes
Different organs (liver, heart, kidneys, thymus, brain) were weighed. Liver was weighed for determination of absolute and relative liver weight.

HISTOPATHOLOGY: yes
Tissues were fixed in 10 % neutral buffered formalin. Tissue specimens were then trimmed, processed through an ascending ethanol series and xylene, embedded in paraffin, and sectioned at 5 μm. One set of tissues was stained with haematoxylin and eosin (H&E). Another set of the pancre as, liver, spleen, kidney, and heart was reacted with Perl’s Prussian blue for Fe, which involves colourless potassium ferrocyanide reacting with ferric ions to create an insoluble blue ferric ferrocyanide (Luna, L.G. et al. (1968), Thompson, S.W. et al. (1966)).*
Pancreas Morphometry: to ensure uniform diameter of sections of equivalent area, pancreatic tissues were adhered to filter paper and fixed in 10 % neutral buffered formalin. Procedures for embedding, sectioning, staining, and evaluating the pancreas were standardized to assure uniformity. Five-micron sections were mounted on positively charged slides and immunostained by using the StreptAvidin peroxidase detection system with DAB (3,3’-diaminobenzidine) as the chromogen substrate and Mayer’s haematoxylin as the counterstain. Sections were incubated with a polyclonal antibody to insulin (1:600 dilution) (Dako Corp.). Cells were counted and areas measured by using the Optimas Image Analysis System. All islets present in a representative section of each pancreas were
evaluated for β-cell number and islet area.

*References:
- Luna LG (1968): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill, New York.
- Thompson SW and Hunt RD (1966): Selected Histochemical and Histopathological Methods. Charles C Thomas, Springfield, Illinois.

Other examinations:

NON-HEME IRON:
- Time schedule for examinations: at study termination (after 90 days of exposure)
- Procedure: non-heme Fe was determined in livers by the bathophenanthroline reaction and expressed as [μg Fe/g liver. Approx. 1 g of each previously blotted liver was weighed and placed in a 50-ml polypropylene centrifuge tube, and distilled, deionized water was added to bring the volume to 15 ml. The tissue was then homogenized for 30 sec with a Polytron. A 3-ml sample of the homogenate was transferred to another 50-ml centrifuge tube, and 10 ml of acid reagent was added (6 M HCI and 1.2 M trichloroacetic acid, 1:1, v/v) and mixed well. This mixture was then heated in an oven at 65 °C for 20 hr, cooled, and centrifuged at 1,500xg for 20 min. Duplicate 0.2-ml aliquots of the supernatant fraction were pipetted into small polypropylene tubes, and 1.8 ml of freshly prepared colour reagent was added, mixed, and incubated for 10 min at room temperature. Absorbance was determined spectrophotometrically at 535 nm, and Fe concentration (μg Fe/ml) was determined by reference to a standard curve. The bathophenanthroline colour reagent (which was protected from light) was prepared by dissolving 62.5 mg bathophenanthroline disulfonic acid and 0.25 ml thioglycolic acid in distilled, deionized water and diluting to 25 ml. The final colour reagent was a solution of the bathophenanthroline colour reagent, saturated sodium acetate (4.5 M), and distilled, deionized water (1:20:20 by volume).

Statistics:

Differences among dietary treatment groups were assessed by 1-way analysis of variance (ANOVA), using the ABstat general linear model program. The Duncan multiple comparison method was used to differentiate among means for variables that were significantly affected by the treatments. Values are expressed as meant- SEM. Correlation coefficients were determined by using Pearson’s productmoment correlation matrix. Mean organ: brain weight ratios were compared by 2-sided Student’s ttests. Immunohistochemical assays and pancreas morphometry data were compared by ANOVA with the Holm’s correction.

Clinical signs:

not specified

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:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Spleen and heart weights were increased in mice.
- Kidney weights were slightly decreased in mice.
- 10000 μg Fe/g: heart:brain weight ratios were increased in mice.
- 5000 and 10000 μg Fe/g: relative liver weights increased statistically significant compared to controls (p<0.001).

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
- 10000 µg Fe/g: in livers of mice at the dose level, moderate Fe deposition in hepatocytes and Kupffer cells, predominantly in the periportal region, was observed (average severity score 3.4). Mice exhibited significant hypertrophy of hepatocytes, primarily in the periportal region.

Pancreas:
- 10000 µg Fe/g: pancreatic acinar cells of mice had an increased content of apical secretory granules (cytoplasmic alteration). Pancreatic morphometry: number of β and δ cells in the pancreatic islets was decreased in mice. However, islet numbers and mean and total islet areas were similar in all mice.

Spleen:
- There were sharply increased numbers of megakaryocytes, unidentifiable blood stem cells, and immature stages of both leukocytes and erythrocytes.

Kidney:
- mice in the 10000 µg Fe/g group exhibited some Fe deposition in the epithelium of the proximal tubules. Mild proliferation of mesangial and/or glomerular epithelial cells was also observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LIVER NON-HEME IRON:
- all dose groups: dose-related increase in liver non-heme iron was observed (exposed groups: 314 - 1981 μg/g; control: 100 ± 5 μg/g).

Please refer to the attached background material (table II) in section "Overall remarks, attachments".

Details on results:

MORTALITY:
- there was no mortality.

BODY WEIGHT AND WEIGHT CHANGES:
- No effects on body weight gain were observed. The 10,000 µg Fe/g dose exerted no inhibitory effect on body weight increase in the mice.

FOOD CONSUMPTION:
- 3500 and 10000 μg Fe/g: food intake was statistically significant decreased compared to controls (p<0.001).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- Spleen:brain weight ratios did not differed significantly between the 10000 µg Fe/g and control groups.

HISTOPATHOLOGICAL FINDINGS:
- Any increase in myocyte volume or number was not detectable in the heart by routine microscopy.

Dose descriptor:

NOAEL

Effect level:

> 3 500 other: μg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

> 425.83 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The effect level [mg/kg bw] was calculated based on the final body weight and feed intake. See the calculations in the field "Any other information on materials and methods incl. tables".

Dose descriptor:

LOAEL

Effect level:

5 000 other: μg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

627.87 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The effect level [mg/kg bw] was calculated based on the final body weight and feed intake. See the calculations in the field "Any other information on materials and methods incl. tables".

Critical effects observed:

not specified

Conclusions:

NOAEL(male mice): > 3500 µg Fe/g diet (equivalent to 425.83 mg Fe/kg bw);
LOAEL (male mice): 5000 µg Fe/g diet (equivalent to 627.87 mg Fe/kg bw)

Whittaker, P. et al. investigated the effects of carbonyl iron in B6C3F1 mice fed at concentrations of 1500, 3500, 5000 and 10000 μg Fe/g in diet for 90 days. At the end of study, food consumption and final body weight were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined. Furthermore, the concentrations of non-heme iron were determined.
According to the authors, no mortality and no effects on body weight gain were observed. There was a dose-related increase in liver non-heme Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. In addition, the relative liver weights were significantly increased in the 5000 and 10000 μg Fe/g dose groups. There was significant hypertrophy of the hepatocytes in mice fed the 10000 μg Fe/g diet. Morphometric evaluation of pancreas showed fewer β-cells.

Deficiencies:
The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low for a statistical analysis. Animals were not observed for clinical signs and cage-side observations were not performed. In addition, animals were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, heamatology or clinical biochemistry were not performed. The histopathological examination was incomplete since trachea, lungs, aorta, gonads, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, peripheral nerve, sternum with bone marrow were not examined. Information about test animals (source, acclimation period, initial body weight) and environmental conditions (photoperiod, temperature, humidity) were insufficiently. The results of the histopathological examination were not shown in tabular form, and the organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reference 8

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low. Animals were not observed for clinical signs, and cage-side observations were not performed. Mice were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, haematology or clinical biochemistry were not performed. Histopathological examination was incomplete. Information about test animals and environmental conditions were insufficient. Organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

C5YSF1 yellow mice were fed to carbonyl iron at concentrations of 1500, 3500, 5000 and 10000 μg Fe/g in diet for 90 days. At the end of study, food consumption and final body weights were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined (liver, pancreas, spleen, heart, kidney and testis epithelium). Furthermore, the concentrations of non-heme iron were determined. Also, the cell proliferation in liver and cells in Langerhans islets were evaluated.

GLP compliance:

not specified

Remarks:

in the publication.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ISP Technologies, Inc. Wayne, NJ

Species:

mouse

Strain:

other: C5YSF1-yellow

Details on species / strain selection:

F1 hybrid mice from the following two crosses were used: C57BL/6NNctr x YS/WffC3Hf/Nctr-Avy -> C5YSF,-yellow (Avy/a)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks old
- Housing: animals were housed in polycarbonate cages, 2 mice/cage
- Diet (ad libitum): AIN-76A diet (source: Dyets, Inc., Bethlehem, PA), which contained (g/kg) casein 200.0, DL-methionine 3.0, cornstarch 150.00, glucose 500.0, fiber Celufil 50.0, AIN-76 mineral mix 35.0, and AIN-76A vitamin mix 10.0 with 50.0 mg menadione/kg and 2.0 g choline bitartrate/kg.
- Water (ad libitum): distilled, deionized water

Route of administration:

oral: feed

Details on route of administration:

Not specified

Vehicle:

not specified

Details on oral exposure:

Not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

35 other: μg Fe/g diet

Remarks:

Control

Dose / conc.:

1 500 other: μg Fe/g diet

Dose / conc.:

3 500 other: μg Fe/g diet

Dose / conc.:

5 000 other: μg Fe/g diet

Dose / conc.:

10 000 other: μg Fe/g diet

No. of animals per sex per dose:

12 males

Control animals:

yes, plain diet

Details on study design:

- Section schedule rationale (if not random): animals were initially divided into 5 groups of approximately equal mean body weight.

Positive control:

Not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule (mortality): not specified

DETAILED CLINICAL OBSERVATIONS: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not specified

FOOD EFFICIENCY: not specified
WATER CONSUMPTION: not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
PLASMA/SERUM HORMONES/LIPIDS: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

Sacrifice and pathology:

GROSS PATHOLOGY: yes
A complete necropsy was performed on each animal.

ORGAN WEIGHT: yes
Different organs (liver, heart, kidneys, thymus, brain) were weighed. Liver was weighed for determination of absolute and relative liver weight.

HISTOPATHOLOGY: yes
Tissues were fixed in 10 % neutral buffered formalin. Tissue specimens were then trimmed, processed through an ascending ethanol series and xylene, embedded in paraffin, and sectioned at 5 μm. One set of tissues was stained with haematoxylin and eosin (H&E). Another set of the pancreas, liver, spleen, kidney, and heart was reacted with Perl’s Prussian blue for Fe, which involves colourless potassium ferrocyanide reacting with ferric ions to create an insoluble blue ferric ferrocyanide (Luna, L.G. et al. (1968), Thompson, S.W. et al. (1966)).*
Pancreas Morphometry: to ensure uniform diameter of sections of equivalent area, pancreatic tissues were adhered to filter paper and fixed in 10 % neutral buffered formalin. Procedures for embedding, sectioning, staining, and evaluating the pancreas were standardized to assure uniformity. Five-micron sections were mounted on positively charged slides and immunostained by using the StreptAvid in peroxidase detection system with DAB (3,3’-diaminobenzidine) as the chromogen substrate and Mayer’s haematoxylin as the counterstain. Sections were incubated with a polyclonal antibody to insulin (1:600 dilution) (Dako Corp.). Cells were counted and areas measured by using the Optimas Image Analysis System. All islets present in a representative section of each pancreas were evaluated for β-cell number and islet area.

*References:
- Luna LG (1968): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill, New York.
- Thompson SW and Hunt RD (1966): Selected Histochemical and Histopathological Methods. Charles C Thomas, Springfield, Illinois.

Other examinations:

NON-HEME IRON:
- Time schedule for examinations: at study termination (after 90 days of exposure)
- Procedure: non-heme Fe was determined in livers by the bathophenanthroline reaction and expressed as [μg Fe/g liver]. Approx. 1 g of each previously blotted liver was weighed and placed in a 50-ml polypropylene centrifuge tube, and distilled, deionized water was added to bring the volume to 15 ml. The tissue was then homogenized for 30 sec with a Polytron. A 3-ml sample of the homogenate was transferred to another 50-ml centrifuge tube, and 10 ml of acid reagent was added (6 M HCI and 1.2 M trichloroacetic acid, 1:1, v/v) and mixed well. This mixture was then heated in an oven at 65 °C for 20 hr, cooled, and centrifuged at 1,500xg for 20 min. Duplicate 0.2-ml aliquots of the supernatant fraction were pipetted into small polypropylene tubes, and 1.8 ml of freshly prepared colour reagent was added, mixed, and incubated for 10 min at room temperature. Absorbance was determined spectrophotometrically at 535 nm, and Fe concentration (μg Fe/ml) was determined by reference to a standard curve. The bathophenanthroline colour reagent (which was protected from light) was prepared by dissolving 62.5 mg bathophenanthroline disulfonic acid and 0.25 ml thio glycolic acid in distilled, deionized water and diluting to 25 ml. The final colour reagent was a solution of the bathophenanthroline colour reagent, saturated sodium acetate (4.5 M), and distilled, deionized
water (1:20:20 by volume).

Statistics:

Differences among dietary treatment groups were assessed by 1-way analysis of variance (ANOVA), using the ABstat general linear model program. The Duncan multiple comparison method was used to differentiate among means for variables that were significantly affected by the treatments. Values are expressed as meant- SEM. Correlation coefficients were determined by using Pearson’s productmoment correlation matrix. Mean organ: brain weight ratios were compared by 2-sided Student’s ttests. Immunohistochemical assays and pancreas morphometry data were compared by ANOVA with the Holm’s correction.

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1500, 3500, 5000 and 10000 µg Fe/g: final body weights were statistically significantly decreased compared to controls (p<0.001). Weight gain was reduced in mice.
Please refer to the attached background material (table IV) in section "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 1500, 3500, 5000 and 10000 µg Fe/g: food intake was statistically significant and dose-related decreased in the mice compared to controls (p<0.001).

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Spleen weights were increased in mice.
- Spleen:brain weight ratios differed significantly between the 10000 µg Fe/g and control groups.
- Heart:brain weight ratios were increased in mice at the 10000 µg Fe/g dose level.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Description (incidence and severity):

Liver:
- 10000 µg Fe/g: moderate Fe deposition in hepatocytes and Kupffer cells was observed in the liver of mice, predominantly in the periportal region. Bile duct inflammation (6/12) and focal hepatocyte necrosis (2/12) were also seen these mice.

Pancreas:
- 10000 µg Fe/g: in the pancreatic morphometry, number of β cells in the pancreatic islets was decreased in mice. Fewer islets were present, and total and mean islet areas were smaller than in the control mice. However, islet numbers and mean and total islet areas were similar in all mice.

Spleen:
- There were sharply increased numbers of megakaryocytes, unidentifiable blood stem cells, and immature stages of both leukocytes and erythrocytes.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LIVER NON-HEME IRON:
- all dose groups: dose-related increase in liver non-heme iron was observed (exposed groups: 887 - 2046 μg/g; control: 36 ± 7 μg/g).
Please refer to the attached background material (table IV) in section "Overall remarks, attachments".

Details on results:

MORTALITY:
- there was no mortality.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- absolute and relative liver weights were not significantly different in the exposed groups compared to controls.

HISTOPATHOLOGICAL FINDINGS:
- Pancreas: pancreas of mice appeared to be unaffected histologically by the Fe treatment.

Dose descriptor:

LOAEL

Effect level:

1 500 other: µg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

152.59 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: The effect level [mg/kg bw] was calculated based on the final body weight and feed intake. See the calculations in the field "Any other information on materials and methods incl. tables".

Critical effects observed:

not specified

Conclusions:

LOAEL (male mice): 1500 µg Fe/g diet (equivalent to 152.59 mg Fe/kg bw)

Whittaker, P. et al. investigated the effects of carbonyl iron in C5YSF1 yellow mice fed at concentrations of 1500, 3500, 5000 and 10000 μg Fe/g in diet for 90 days. At the end of study, food consumption and final body weight were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined. Furthermore, the concentrations of non-heme iron were determined.
According to the authors, no mortality was observed. At all doses of Fe above the control, food intake and body weight gain was reduced in mice. In all animals, there was a dose-related increase in liver non-heme Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. Morphometric evaluation of pancreas showed fewer β-cells in mice. There were fewer islets in the yellow C5YSF1 mice, and total and mean islet areas were smaller than in the control mice.

Deficiencies:
The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low for a statistical analysis. Animals were not observed for clinical signs and cage-side observations were not performed. In addition, animals were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, heamatology or clinical biochemistry were not performed. The histopathological examination was incomplete since trachea, lungs, aorta, gonads, oesaphagus, stomach, duodenum, jejunm, ileum, caecum, colon, rectum, urinary bladder, peripheral nerve, sternum with bone marrow were not examined. Information about test animals (source, acclimation period, initial body weight) and environmental conditions (photoperiod, temperature, humidity) were insufficiently. The results of the histopathological examination were not shown in tabular form, and the organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reference 9

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low. Animals were not observed for clinical signs, and cage-side observations were not performed. Mice were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, haematology or clinical biochemistry were not performed. Histopathological examination was incomplete. Information about test animals and environmental conditions were insufficient. Organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

C5YSF1 black mice were fed to carbonyl iron at concentrations of 1500, 3500, 5000 and 10000 μg Fe/g in diet for 90 days. At the end of study, food consumption and final body weights were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined (liver, pancreas, spleen, heart, kidney and testis epithelium). Furthermore, the concentrations of non-heme iron were determined. Also, the cell proliferation in liver and cells in Langerhans islets were evaluated.

GLP compliance:

not specified

Remarks:

in the publication.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ISP Technologies, Inc. Wayne, NJ

Species:

mouse

Strain:

other: C5YSF1 black

Details on species / strain selection:

F1 hybrid mice from the following two crosses were used: C57BL/6NNctr x YS/WffC3Hf/Nctr-Avy -> C5YSF black (Avy/a)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks old
- Housing: animals were housed in polycarbonate cages, 2 mice/cage
- Diet (ad libitum): AIN-76A diet (source: Dyets, Inc., Bethlehem, PA), which contained (g/kg) casein 200.0, DL-methionine 3.0, cornstarch 150.00, glucose 500.0, fiber Celufil 50.0, AIN-76 mineral mix 35.0, and AIN-76A vitamin mix 10.0 with 50.0 mg menadione/kg and 2.0 g choline bitartrate/kg.
- Water (ad libitum): distilled, deionized water

Route of administration:

oral: feed

Details on route of administration:

Not specified

Vehicle:

not specified

Details on oral exposure:

Not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

35 other: μg Fe/g diet

Remarks:

Control

Dose / conc.:

1 500 other: μg Fe/g diet

Dose / conc.:

3 500 other: μg Fe/g diet

Dose / conc.:

5 000 other: μg Fe/g diet

Dose / conc.:

10 000 other: μg Fe/g diet

No. of animals per sex per dose:

12 males

Control animals:

yes, plain diet

Details on study design:

- Section schedule rationale (if not random): animals were initially divided into 5 groups of approximately equal mean body weight.

Positive control:

Not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule (mortality): not specified

DETAILED CLINICAL OBSERVATIONS: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not specified

FOOD EFFICIENCY: not specified
WATER CONSUMPTION: not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
PLASMA/SERUM HORMONES/LIPIDS: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

Sacrifice and pathology:

GROSS PATHOLOGY: yes
A complete necropsy was performed on each animal.

ORGAN WEIGHT: yes
Different organs (liver, heart, kidneys, thymus, brain) were weighed. Liver was weighed for determination of absolute and relative liver weight.

HISTOPATHOLOGY: yes
Tissues were fixed in 10 % neutral buffered formalin. Tissue specimens were then trimmed, processed through an ascending ethanol series and xylene, embedded in paraffin, and sectioned at 5 μm. One set of tissues was stained with haematoxylin and eosin (H&E). Another set of the pancreas, liver, spleen, kidney, and heart was reacted with Perl’s Prussian blue for Fe, which involves colourless potassium ferrocyanide reacting with ferric ions to create an insoluble blue ferric ferroc yanide (Luna, L.G. et al. (1968), Thompson, S.W. et al. (1966)).*
Pancreas Morphometry: to ensure uniform diameter of sections of equivalent area, pancreatic tissues were adhered to filter paper and fixed in 10 % neutral buffered formalin. Procedures for embedding, sectioning, staining, and evaluating the pancreas were standardized to assure uniformity. Five-micron sections were mounted on positively charged slides and immunostained by using the StreptAvid in peroxidase detection system with DAB (3,3’-diaminobenzidine) as the chromogen substrate and Mayer’s haematoxylin as the counterstain. Sections were incubated with a polyclonal antibody to insulin (1:600 dilution) (Dako Corp.). Cells were counted and areas measured by using the Optimas Image Analysis System. All islets present in a representative section of each pancreas were
evaluated for β-cell number and islet area.

*References:
- Luna LG (1968): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill, New York.
- Thompson SW and Hunt RD (1966): Selected Histochemical and Histopathological Methods. Charles C Thomas, Springfield, Illinois.

Other examinations:

NON-HEME IRON:
- Time schedule for examinations: at study termination (after 90 days of exposure)
- Procedure: non-heme Fe was determined in livers by the bathophenanthroline reaction and expressed as [μg Fe/g liver]. Approx. 1 g of each previously blotted liver was weighed and placed in a 50-ml polypropylene centrifuge tube, and distilled, deionized water was added to bring the volume to 15 ml. The tissue was then homogenized for 30 sec with a Polytron. A 3-ml sample of the homogenate was transferred to another 50-ml centrifuge tube, and 10 ml of acid reagent was added (6 M HCI and 1.2 M trichloroacetic acid, 1:1, v/v) and mixed well. This mixture was then heated in an oven at 65 °C for 20 hr, cooled, and centrifuged at 1,500xg for 20 min. Duplicate 0.2-ml aliquots of the supernatant fraction were pipetted into small polypropylene tubes, and 1.8 ml of freshly prepared colour reagent was added, mixed, and incubated for 10 min at room temperature. Absorbance was determined spectrophotometrically at 535 nm, and Fe concentration (μg Fe/ml) was determined by reference to a standard curve. The bathophenanthroline colour reagent (which was protected from light) was prepared by dissolving 62.5 mg bathophenanthroline disulfonic acid and 0.25 ml thioglycolic acid in distilled, deionized water and diluting to 25 ml. The final colour reagent was a solution of the bathophenanthroline colour reagent, saturated sodium acetate (4.5 M), and distilled, deionized water (1:20:20 by volume).

Statistics:

Differences among dietary treatment groups were assessed by 1-way analysis of variance (ANOVA), using the ABstat general linear model program. The Duncan multiple comparison method was used to differentiate among means for variables that were significantly affected by the treatments. Values are expressed as meant- SEM. Correlation coefficients were determined by using Pearson’s productmoment correlation matrix. Mean organ: brain weight ratios were compared by 2-sided Student’s ttests. Immunohistochemical assays and pancreas morphometry data were compared by ANOVA with the Holm’s correction.

Clinical signs:

not specified

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:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 10000 µg Fe/g: absolute and relative liver weights were statistically significant increased compared to controls (p<0.02).
- Spleen and heart weights were increased in mice.
- Spleen:brain weight ratios differed significantly between the 10000 µg Fe/g and control groups.
- 10000 µg Fe/g: heart:brain weight ratios were increased in mice.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Liver:
- 10000 µg Fe/g: in livers of mice moderate Fe deposition in hepatocytes and Kupffer cells, predominantly in the periportal region, was observed.

Spleen:
- There were sharply increased numbers of megakaryocytes, unidentifiable blood stem cells, and immature stages of both leukocytes and erythrocytes.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LIVER NON-HEME IRON:
- all dose groups: dose-related increase in liver non-heme iron was observed (exposed groups: 253 - 2021 μg/g; control: 45.2 ± 3 μg/g).
Please refer to the attached background material (table III) in section "Overall remarks, attachments".

Details on results:

FOOD CONSUMPTION AND COMPOUND INTAKE:
- food intake was not affected by supplemental Fe.

BODY WEIGHT AND WEIGHT CHANGES:
- No effects on body weight gain were observed. The 10000 µg Fe/g dose exerted no inhibitory effect on body weight increase in the mice.

HISTOPATHOLOGICAL FINDINGS:
- Pancreas: pancreas of mice appeared to be unaffected histologically by the Fe treatment. No effect on the pancreatic islets morphometry was observed.
- Heart: any increase in myocyte volume or number was not detectable by routine microscopy.

Dose descriptor:

NOAEL

Effect level:

> 5 000 other: µg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

> 604.4 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The effect level [mg/kg bw] was calculated based on the final body weight and feed intake. See the calculations in the field "Any other information on materials and methods incl. tables".

Dose descriptor:

LOAEL

Effect level:

10 000 other: µg Fe/g diet

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

1 300.78 other: mg Fe/kg bw

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The effect level [mg/kg bw] was calculated based on the final body weight and feed intake. See the calculations in the field "Any other information on materials and methods incl. tables".

Critical effects observed:

not specified

Conclusions:

NOAEL (male mice): > 5000 μg Fe/g diet (equivalent to 604.40 mg Fe/kg bw);
LOAEL (male mice): 10,000 μg Fe/g diet (equivalent to 1300.78 mg Fe/kg bw)

Whittaker, P. et al. investigated the effects of carbonyl iron in C5YSF1 black mice fed at concentrations of 1500, 3500, 5000 and 10000 μg Fe/g in diet for 90 days. At the end of study, food consumption and final body weight were recorded. A full necropsy of all animals was performed, organs were weighed and histopathologically examined. Furthermore, the concentrations of non-heme iron were determined.
According to the authors, no mortality was observed. No effects on food intake or body weight gain were observed. The liver weights were increased in the 10000 10000 µg Fe/g dose group. In all animals, there was a dose-related increase in liver non-heme Fe, and the Fe was stored in hepatocytes predominantly in the periportal region.

Deficiencies:
The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low for a statistical analysis. Animals were not observed for clinical signs and cage-side observations were not performed. In addition, animals were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, heamatology or clinical biochemistry were not performed. The histopathological examination was incomplete since trachea, lungs, aorta, gonads, oesaphagus, stomach, duodenum, jejunm, ileum, caecum, colon, rectum, urinary bladder, peripheral nerve, sternum with bone marrow were not examined. Information about test animals (source, acclimation period, initial body weight) and environmental conditions (photoperiod, temperature, humidity) were insufficiently. The results of the histopathological examination were not shown in tabular form, and the organ weights of heart, kidneys, brain and thymus were not presented in the publication.

Reference 10

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose toxicity study with the reproductive/ developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2011 - 08 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD test guidelines, and in compliance with GLP, so the data is considered reliable without restriction.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996-03-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of toxic and reproductive change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available in this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approx. 72 days
- Weight at study initiation: 348 - 399 g (males), 230 - 287 g (females)
- Housing: Polycarbonate cages with either stainless steel grid floors during mating, and solid poly carbonate floors during other phases of testing.
- Diet (e.g. ad libitum): ad libitum / free access, except overnight before routine blood sampling
- Water (e.g. ad libitum): ad libitum / free access
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 26 October 2011 (Treament commenced) To: 12 December 2011 (Last date of necropsy for main phase females)

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

other: 1% (w/v) aqueous methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared weekly, up to seven days in advance of the first day of dosing and were stored refrigerated (2-8 °C).

VEHICLE
- Concentration in vehicle: 10 - 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation samples were analysed by Atomic Absorption Spectrometry, to determine Iron in the samples.

Prior to the start of treatment, the analytical method was validated with respect to specificity, limit of detection, linearity of detector response over the calibration range, precision of measurement at the lowest and highest calibration standards, and the accuracy and precision of the method, by the determination of six procedural recoveries at 1 mg/mL and 100 mg/mL. A stability tiral was also performed; formulations were found to be stable and homogenous for up to 2 hours at ambient temperature with paddle stirring, and following 15 days' refrigerated storage.

Samples from all formulations prepared in the first and last weeks of the study were analysed; the test material concentrations were found to be within acceptable limits, confirming accurate preparation.

Duration of treatment / exposure:

Main phase males and toxicity phase females were dosed for five consecutive weeks.
Main phase females were treated daily for two weeks before pairing, throughout mating, gestation and until day 6 of lactation.

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

Main phase - 10 animals per sex per dose
Toxicity phase - 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were decided in a 7-day preliminary study in rats, conducted at the same laboratory - refer to "7 Day rangefinding_Huntingdon Life Sciences, 2011 (FGE0026)". In the current dose range finder study, groups of three male and three female Crl: CD(SD) rats were administered with iron phosphorous in 1 % w/v aqueous methylcellulose via gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats were exposed daily to the test substance for 7 days. No test item-related effects were observed for clinical signs, mortality, body weight gain, food consumption, organ weights and gross pathology. Therefore, the oral administration of Fe3P to Crl:CD(SD) rats at doses up to 1000 mg/kg/day was well tolerated, causing no adverse response to treatment. In the absence of any toxicological findings after seven days of treatment, doses up to 1000 mg/kg/day are considered suitable for use on the planned OECD 422 study (FGE0027). Please refer to the study record w_O'Halloran_2011_FeP3 at IUCLID section 7.5.1.
- Post-exposure recovery period in satellite groups: Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Prior to the start of treatment, and at least weekly for males and toxicity phase females. Main phase females were observed weekly before pairing, and on days 0, 6, 13 and 20 after mating, and days 1 and 7 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations: Main phase males and toxicity phase females were weighed on day 0 of treatment, then weekly and beofre necropsy. Main phase females were weighed on day 0 of treatment and weekly until mating was detected, and days 0, 6, 13 and 20 after mating, and on days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 5 of treatment for 5 of the main phase males and for the toxicity phase females.
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: Yes
- How many animals: As above
- Parameters checked: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential Leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)), Platelet count (Plt).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: conducted at the same time and using the same animals as Haematology, above.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength measured in the first five males for the main phase, and all five females of the toxicity phase during week 5 of treatment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights recorded at necropsy (F0 animals; L&R – Bilateral organs weighed individually): Adrenals, Prostate, Brain, Seminal vesicles with coagulating gland, Epididymides (L&R), Spleen, Heart, Testes (L&R), Kidneys, Thymus, Liver, Uterus (including cervix and oviducts), Ovaries (L&R)
- Gross necropsy, fixation (F0 animals): Adrenals, Pituitary, Brain, Prostate, Caecum, Rectum, Colon, Sciatic nerves (only one per adult animal processed for examination), Duodenum, Seminal vesicles with coagulation glands, Epididymides, Skeletal muscle (only one per adult animal processed for examination), Eyes, Spinal cord, Heart, Spleen, Ileum, Sternum with marrow, Jejunum, Stomach, Kidneys, Testes, Liver, Thymus, Lungs, Thyroid with parathyroids, Lymph nodes (axillary, mesenteric), Trachea, Urinary bladder, Oesophagus, Uterus (including cervix and oviducts), Ovaries, Vagina, Peyer’s patch

HISTOPATHOLOGY: Yes
- Tissues: Adrenal (cortex and medulla), Brain (cerebellum, cerebrum and pons), Heart (included auricular and ventricular regions), Kidneys (included cortex, medulla and papilla regions), Liver (section from two main lobes), Lungs (section from two major lobes, to include bronchi), Ovaries (qualitative evaluation of one section from each ovary), Seminal vesicles (included coagulating glands in section), Spinal cord (transverse and longitudinal section at the cervical, lumbar and thoracic levels), Sternum (included bone marrow), Stomach (included keratinised, glandular and antrum in sections), Thyroid (included parathyroids in section where possible), Uterus (uterine body with cervix section and oviducts)

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, organ weight, litter size, survival indices and clinical pathology data:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead. For grip strength, motor activity survival indices and clinical pathology, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During the study there were no signs that were considered to be related to the administration of Fe3P and no unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain for males during the five weeks of treatment was unaffected by the administration of Fe3P.
Bodyweight gain for females during the first two weeks of treatment was considered to be unaffected by the administration of Fe3P. Toxicity phase females had lower bodyweight gain between weeks three and five of treatment (max 47% of Control) along with a lower overall bodyweight gain over the five weeks of treatment (max 60% of Control). However, there was no dose response and a relationship to treatment is uncertain.
Bodyweight gain for gestating females was considered the be unaffected by the administration of Fe3P, however lactating females given Fe3P at 300 and 1000 mg/kg/day had marginally lower bodyweight gain between days one and seven of lactation (max 82% of Control)

FOOD CONSUMPTION
Food consumption for males was considered to be unaffected by the administration of Fe3P.
During the first two weeks of treatment female food consumption was considered to be unaffected by the administration of Fe3P. Toxicity phase females showed some intergroup variation in food consumption during week 3 of treatment. During Weeks four and five food consumption was marginally lower in those animals given Fe3P (94% and 90% of Controls respectively). However, there was no dose response and a relationship to treatment is uncertain.
Food consumption for gestating and lactating females, with the exception of females given 1000 mg/kg/day, was considered to be unaffected by the administration of Fe3P. Females given 1000 mg/kg/day had marginally lower food consumption over the seven days of lactation when compared to Controls.

HAEMATOLOGY
Haematological parameters in both males and females in Week 5 of treatment were considered to be unaffected by the administration of Fe3P. Males given 300 or 1000 mg/kg/day had slightly low haematocrit and haemoglobin concentrations which attained statistical significance when compared to control, but there were no similar effects in females.

CLINICAL CHEMISTRY
Blood chemistry parameters in males and females in Week 5 of treatment were considered to be unaffected by the administration of Fe3P.

ORGAN WEIGHTS
There was no effect of treatment on organ weights for males receiving Fe3P.
Intergroup variability in uterine weight did not appear to follow any dose related pattern and may relate to differences in the stage of the oestrous cycle at necropsy.

GROSS PATHOLOGY
The macroscopic examination performed after 5 weeks of treatment revealed no test substance related lesions.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes.

HISTOPATHOLOGY
Microscopic examination was performed on the first five Main phase males killed at scheduled termination and Toxicity phase females in the Control and high dose group (1000 mg/kg/day).
In all the tissues examined, there was no evidence of changes considered related to the treatment with the test compound.
The seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage abnormalities were noted.

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Conclusions:

In the current combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, iron phosphorous (Fe3P) in 1 % (w/v) aqueous methylcellulose was administered via gavage 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 five consecutive weeks or daily for two weeks before pairing throughout mating, gestation and until lactation day 6 for males and females, respectively (main phase groups). A vehicle control group was run concurrently. In addition, groups of 5 females were administered the substance at dose levels of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day via gavage for five consecutive days and these females were not mated (toxicity phase groups).

During the observations of the parental animals, no test item-related effects were observed for clinical signs, mortality, body weight gain, food consumption, haematology, clinical biochemistry, neurobehavioural examinations, organ weights, gross pathology and histopathology.

Based on the results from this study, for general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.

Reference 11

Endpoint:

repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 September 2011 - 02 December 2011

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study is a preliminary range-finding study, conducted in preparation for a 28-day repeat oral dose toxicity screen and reproductive / developmental toxicity screening study. Although the methodology employed was sound and the study was well documented, the study was not formally conducted in compliance with GLP (rather, only in the spirit of GLP), and should not be considered as a standalone study or outwith the context of the main study (Huntingdon Life Sciences study FGE0027).

Qualifier:

no guideline followed

GLP compliance:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance by regulatory agencies. The Crl:CD(SD) strain was used because of the background data available in this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approx. 10 weeks
- Weight at study initiation: 333 - 384 g (males) and 238 - 266 g (females)
- Housing: 3 rats of the same sex per cage, ploycarbonate cage with stainless steel lid
- Diet (e.g. ad libitum): Ad libitum (supply not restricted)
- Water (e.g. ad libitum): Ad libitum (supply not restricted)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 40 - 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 05 October 2011 To:12 October 2011

Route of administration:

oral: gavage

Details on route of administration:

The route of administration was oral gavage as to simulate the conditions of potential human exposure.

Vehicle:

other: 1% w/v Aqueous Methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Freshly prepared each day and administered within 2 hours of preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% (w/v) Aqueous methylcellulose
- Concentration in vehicle: 10, 30, or 100 mg/mL.
- Amount of vehicle (if gavage):10 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

3

Control animals:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Animals and their cages observed for evidence of ill-health at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, immediately after dosing on return of the animal to its cage, on completion of dosing of each group, between one and two hours after completion of dosing of all groups, as late as possible in the working day. In addition, a more detailed physical examination was performed on each animal on Days 1, 4 and 8 of study to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: during acclimatisation, and on days 1, 4, and 8

FOOD CONSUMPTION:
- Food consumption recorded from 3 days before start of treatment to the day before the start of treatment (days -3 to -1), then from days 1-3 and 4-7.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by visual observation. No effect was observed and consequently
quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Macroscopic pathology conducted on all animals. Thoracic and abdominal cavities opened, cranial cavity opened only if observations indicated possible neurotoxic action.
- Organ weights recorded for Liver, Spleen, Kidneys, and known or identified target organs (identified during the course of treatment).

HISTOPATHOLOGY: No

Statistics:

No statistical analysis was performed on this study.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Dose descriptor:

dose level: Maximum dose level to be used in subsequent OECD422 study

Effect level:

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

Based on:

test mat.

Sex:

male/female

Conclusions:

In the current dose range finder study, groups of three male and three female Crl: CD(SD) rats were administered with iron phosphorous in 1 % w/v aqueous methylcellulose via gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats were exposed daily to the test substance for 7 days.

No test item-related effects were observed for clinical signs, mortality, body weight gain, food consumption, organ weights and gross pathology. Therefore, the oral administration of Fe3P to Crl:CD(SD) rats at doses up to 1000 mg/kg/day was well tolerated, causing no adverse response to treatment.

In the absence of any toxicological findings after seven days of treatment, doses up to 1000 mg/kg/day are considered suitable for use on the planned OECD 422 study (FGE0027).

Reference 12

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The following experimental and recording deficiencies can be reported for the publication: test substance is a mixture, therefore, it is unclear, if the observed effects were due to the iron content of the mixture alone; mixture has a low pH (3.7), which might cause the observed effects in the intestine; exposure duration was not clearly described; exposure duration to long; clinical signs were not fully described; detailed clinical examinations were not fully described; historical control data and individual data missing.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996 - 03 - 22

Deviations:

yes

Remarks:

prolonged exposure during pre-mating phase as described according to guideline at least 14 days premating exposure in the study 12 weeks premating exposure

Principles of method if other than guideline:

The OECD guideline 408 (1998) was also followed during the conducted of study.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Source: Akzo Nobel Industrial Chemicals, Inc. (Amersfoort, The Netherlands)

- Stability and storage: the product was in complete aqueous solution and did not tend to settle out or precipitate. It was stated to be stable through the time frame of the study, and stored at room temperature in a sealed tin foil-covered container protected from light.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Horst, the Netherlands)
- Females non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: males 307 - 351g; females 223 - 251g
- Housing: housed in Macrolon plastic cages (18 cm height, Bio-Services, Uden, the Netherlands) bedding material: sterilized sawdust (Litalabo SPPS, Argenteuill, France) and paper (Enviro-dri, Wonham Mill Ltd., Surrey, United Kingdom) as cage enrichment.
- Acclimatization and premating periods: rats were housed in groups of 5 in Macrolon Type MIV cages
- Cohabitation period (mating): 1 male / 1 female of the same group were housed together in Makrolon Type MIII cages
- Post mating: males were housed as in the premating phase while females were individually housed in their respective Makrolon Type MIII cages.
- During locomotor activity monitoring: all animals were individually housed in Hi-temp polycarbonate cages (Ancare Corp., Bellmore, N.Y., U.S.A.) without cage enrichment or bedding material.

- Diet (ad libitum): pelleted diet SM R/M (SSNIFF Spezialdiaeten GmbH, Soest, Germany)
- Water (ad libitum): municipal tap water

- Acclimatization period: length of period not stated, but acclimatization was done

ENVIRONMENTAL CONDITIONS
- Temperature: 17.6°C to 22.8 ◦C
- Humidity: 31% to 81%
- Air changes: approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

not specified

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
FemTA was used as supplied

Dosing was achieved by the applied volume. Dosing volume (mL/kg bw) was calculated based on: (dose level [g/kg]/density [g/cm3]×100/35 [purity adjustment]) where the latest body weight measurements were used. Controls received the same dosing volume (distilled water) as the high-dose group.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

- males: 12 weeks pre-mating, during mating, and up to the day prior to scheduled sacrifice during the post-mating period (total of 90/91 days)
- females (pregnant/littered): 12 weeks pre-mating, during mating, during pregnancy (gestation) and during at least 4 d of lactation (ranging from a total period of 104 to 109 days)
- females (mated but not pregnant): 12 weeks pre-mating, during mating, during the post-mating period (ranging from a total period of 104 to 109 days)

Frequency of treatment:

once daily, 7 days per week

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

equivalent to 20 mg/kg bw/d Fe(III)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 40 mg/kg bw/d Fe(III)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 80 mg/kg bw/d Fe(III)

No. of animals per sex per dose:

treatment groups 10 male + 10 female
control group 10 male + 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a preliminary 10-d dosing range finding study, no evidence of toxicity was noted at doses of up to 2000 mg/kg bw/d.

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: - clinical signs at least once daily immediately after dosing
- mortality/viability at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly intervals outside of the home cage in a standard arena.
Any abnormal findings were recorded with respect to symptom and graded according to a fixed scale.

BODY WEIGHT: Yes
- Time schedule:
males: first day of treatment and weekly thereafter
females: first day of treatment and weekly thereafter gestation on days 0, 4, 7, 11, 14, 17, and 20 and during lactation days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule: weekly except during the mating phase. Following mating, food consumption by females was measured on the same days as body weight measurements were recorded.

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: not specified

WATER CONSUMPTION: Yes
- Time schedule for examination: subjective evaluation only

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimation period and week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: isoflurane (Abbott BV, Hooffddrop, the Netherlands) in nitrous oxide/oxygen (Air Products, Amsterdam, the Netherlands) anaesthesia.
- Animals fasted: Yes, overnight
- How many animals: 8-10 per group
- Parameters checked: activated partial thromboplastin time, APTT; haematocrit, HCT; mean corpuscular haemoglobin, MCH; mean corpuscular haemoglobin concentration, MCHC; mean corpuscular volume, MCV; mean platelet volume, MPV; platelet; PT, prothrombin time, PLT; red blood cell count, RBC; reticulocyte, RET; red blood cell count, RBC; red cell distribution width, RDW; white blood cells, WBC; neutrophil (%WBC); lymphocytes (%WBC); monocytes (%WBC); eosinophiles (%WBC); basophiles (%WBC); haemoglobin

CLINICAL CHEMISTRY: Yes;
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: isoflurane (Abbott BV, Hooffddrop, the Netherlands) in nitrous oxide/oxygen (Air Products, Amsterdam, the Netherlands) anaesthesia.
- Animals fasted: Yes, overnight
- How many animals: all males and 9-10 females per group
- Parameters checked: alanine aminotransferase, ALAT; aspartate aminotransferase, ASAT; alkaline phosphatase, ALP; total protein; albumin; total bilirubin; urea; creatinine; glucose; cholesterol; bile acids; sodium; potassium; chloride; calcium; inorganic phosphate

PLASMA/SERUM HORMONES/LIPIDS: not specified

URINALYSIS: not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observations:
- Time schedule: males during week 13; females near the end of the lactation period
- Dose groups that were examined: all animals
- Function tested: hearing ability, papillary reflex, static righting reflex, and grip strength.

Locomotor activity:
- Time schedule: males during week 12; females near the end of the lactation period
- Dose groups that were examined: all animals
- Function tested: all animals were caged individually and monitored under normal light conditions for activity by a computerized monitoring system (Kinder Scientific LLC, Poway, Calif., U.S.A.). Total movements and ambulations were recorded. Ambulations represent movements characterized by a relocation of the entire body position, such as walking. Total movements represent all movements made by the animals, including ambulations, but also smaller or finer movements like grooming, weaving, or movements of the head.

IMMUNOLOGY: not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were fasted overnight and sacrificed Next, the animals were subject to macroscopic evaluation. The numbers of former implantation sites and corpora lutea were recorded. Furthermore, the following organs weights were recorded adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus, prostate, seminal vesicles, and thyroid gland

HISTOPATHOLOGY: Yes
The following organs and tissues were fixed in 10% buffered formalin and preserved at necropsy from all vehicle control- and high-dose rats:
adrenal glands, aorta, brain, cecum, colon, cervix, clitoral gland, coagulation gland, duodenum, female mammary area, femur, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular and mesenteric), oesophagus, ovaries, pancreas, pituitary gland, Peyer’s patches, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, salivary gland, skeletal muscle, skin, tongue, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid and parathyroid, trachea, urinary bladder, uterus, and vagina, as well as all gross lesions.

Additional slides were prepared from the cecum, colon, and rectum of low- and mid-dose groups to assess a potential treatment-related effect. The samples were embedded in paraffin wax, sectioned (2 to 4 μm), and stained with haematoxylin and eosin (Klinipath).

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett test (Dunnett 1955) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel test was applied if the data could not be assumed to follow a normal distribution. The Fisher’s exact test was applied to frequency data. All tests were 2-sided and in all cases P < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances.

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Following statistically significant changes were considered treatment-related.
1) Males:
- 2000 mg/kg bw/d: higher white blood cell (WBC, P<0.01) counts, higher relative neutrophil counts (P<0.05) and lower relative lymphocytes counts (P<0.05) compared to control were noted.

2) Females:
- 2000 mg/kg bw/d: higher relative eosinophil counts (P<0.05) compared to control were observed.

Please also refer to the field “Attached background material”.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Compared to control, following statistically significant changes were considered treatment-related.
1) Males:
- 1000 and 2000 mg/kg bw/d: increased blood urea nitrogen (BUN, P<0.01) and bile acid levels (P<0.01)
- 2000 mg/kg bw/d: increased alanine aminotransferase (ALAT, P<0.01), reduced sodium (P<0.05) and chloride (P<0.01) concentrations

2) Females:
- 2000 mg/kg bw/d: increased blood urea nitrogen (BUN, P<0.05) and reduced chloride (P<0.05) concentrations.

Please also refer to the field “Attached background material”.

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Compared to control, following statistically significant changes were considered treatment-related.
1) Males:
- 500, 1000 and 2000 mg/kg bw/d: higher absolute (P<0.05) and relative kidney weight (P<0.01)
- 2000 mg/kg bw/d: higher absolute (P<0.05) and relative liver weight (P<0.01)

2) Females:
- 1000 and 2000 mg/kg bw/d: higher relative kidney weight (P<0.01)
- 2000 mg/kg bw/d: higher absolute kidney weight (P<0.01)

Alterations in kidney weight were clearly treatment-related, and, given the magnitude, were considered adverse at the 2000 mg/kg body weight/d dose level. Please also refer to the field “Attached background material”.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compared to control, following statistically significant changes were considered treatment-related.
Males and Females:
- 1000 and 2000 mg/kg bw/d: increase in both incidence and severity of neutrophilic infiltrates, acute inflammation, goblet/ epithelial cell hyperplasia, foci of brown pigment and/or oedema in the rectum, colon and cecum was observed.
Please also refer to the field “Attached background material”.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS:
1) Males and Females:
- 500, 1000 and 2000 mg/kg bw/d: the test item appeared to be well tolerated. Dark coloured faeces were observed from week 2 of the reproduction phase onwards, week 7 of the premating phase onwards, and week 5 on the pre-mating phase onwards, respectively. This was due to the staining properties of the material (FemTA). Salivation was also noted shortly after dosing. This was likely a response to the taste of the substance rather than due to a systemic reaction.

MORTALITY
1) Males and Females:
- 500, 1000 and 2000 mg/kg bw/d: no mortality/morbidity were noted in any of the animals of both sex throughout the experimental test period
2) Females:
- 0 mg/kg bw/d: One female was killed in extremis on day 70 of the premating period. No cause of death could be determined.

BODY WEIGHT AND WEIGHT CHANGES
1) Males:
- 500 mg/kg bw/d: body weights and body weight gain of males were similar to control levels
- 1000 and 2000 mg/kg bw/d: from week 8 and 3 onwards respectively, achieving a level of statistical significance (P<0.01).
2) Females:
- 500 mg/kg bw/d: body weights and body weight gain of females were similar to control levels
- 1000 mg/kg bw/d: statistically significant (P<0.05) lower body weight gain on day 4 of the lactation phase.
- 2000 mg/kg bw/d: statistically significant (P<0.05) lower body weight gain in week 5 of the premating phase
All of the changes were of small magnitude (not exceeding 10%) and were considered of minimal toxicological significance. Please also refer to the field “Attached background material”

FOOD CONSUMPTION
1) Males and Females:
- 500, 1000, and 2000 mg/kg bw/d: No statistically significant changes in food consumption were recorded at any time during the study at any dose level compared to the control.

WATER CONSUPTION
1) Males and Females:
- 500, 1000, and 2000 mg/kg bw/d: Based on a subjective evaluation, there were no discernible differences between the treated groups and controls with respect to water consumption.

OPHTHALMOLOGICAL FINDING
1) Males and Females:
- 500, 1000 and 2000 mg/kg bw/d: The ophthalmological examinations (data not shown) revealed no effect of FemTA treatment.

HAEMATOLOGICAL FINDINGS
1) Males
- 1000 and 2000 mg/kg bw/d: higher mean corpuscular haemoglobin of males at 1000 (P<0.05) and 2000 mg/kg (P<0.01) occurred in the absence of concurrent changes in red blood cell parameters. Also, the means were within the range considered normal for rats of this age and strain.
- 2000 mg/kg bw/d: lower red blood cell counts (P<0.05) and lower platelet (PT) counts (P<0.05) were considered to be within normal ranges for rats of this age and strain. It was not considered of biological importance since the opposite effect (that is, an increase in PT) would be expected in case of target organ toxicity.
2) Females
- 500, 1000 and 2000 mg/kg bw/d: with increasing dose a tendency to decreasing lymphocyte and increasing neutrophil counts was noted in treated females.
3) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: compared to the controls, the results showed no statistical significant changes for: activated partial thromboplastin time, APTT; haematocrit, HCT; mean corpuscular haemoglobin concentration, MCHC; mean corpuscular volume, MCV; mean platelet volume, MPV; prothrombin time, PLT; reticulocyte, RET; red cell distribution width, RDW; monocytes (%WBC); basophiles (%WBC); haemoglobin
Please also refer to the field “Attached background material”.

CLINICAL BIOCHEMESTRY FINDING
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: compared to the controls, the results showed no statistical significant changes for: aspartate aminotransferase, ASAT; alkaline phosphatase, ALP; total protein; albumin; total bilirubin; creatinine; glucose; cholesterol; potassium; calcium; inorganic phosphate
Please also refer to the field “Attached background material”.

BEHAVIOUR (FUNCTIONAL FINDINGS)
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: Hearing ability, pupillary reflex, static righting reflex, and grip strength were similar to the control group in all treated animals. In the motor activity assessment all groups showed a similar habituation profile with high activity in the first testing interval that decreased over the duration of the test period (data not shown). In high-dose females, lower total movement and, in all treated females, lower ambulation counts, were considered to have occurred due to slightly lower total movements/ambulation counts halfway through the measurement period (that is, during the 6th and 7th 5-min interval of the twelve 5-min intervals measured). Since this was of a temporary nature it was considered not to represent a change of toxicological significance.

ORGAN WIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
1) Males
- 1000 mg/kg bw/d: relative testes weights (P<0.05) were increased compared to control.
- 2000 mg/kg bw/d: relative spleen weights (P<0.01) were increased. Changes occurred in the absence of clear dose–response relationships and with respect to the findings in 2000 mg/kg bw/d dose males, were largely attributable to lower terminal body weights as evidenced by the lack of any statistically significant effects on absolute organ weight of the spleen.
2) Females
- 500 mg/kg bw/d: absolute thyroid weight (P<0.05) was reduced
- 1000 mg/kg bw/d: brain weights (P<0.05) were increased. Changes occurred in the absence of clear dose–response relationships.
3) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: the results showed no statistical significant changes for compared to control for the following organs: adrenal glands, epididymites, heart ovaries, thymus, uterus, prostate and seminal vesicles.
Please also refer to the field “Attached background material”.

GROSS PATHOLOGICAL FINDINGS
1) Females
- 500, 1000 and 2000 mg/kg bw/d: at necropsy black contents in the gastrointestinal tract, primarily the cecum and colon, were observed in most treated females, but not in males of any dose group.

2) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: all other findings were within the normal range of variation for rats of this age and strain.

HISTOPATHOLOGICAL FINDINGS (NON-NEOPLASTIC)
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: the remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain and included, for example, grey–white foci in the lungs, tan foci on the preputial glands, reduced size of preputial glands or testes/epididymites, pelvic dilatation of the kidneys, and enlarged lymph node. None of these lesions were related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

equivalent to Fe(III) 20 mg/kg bw/day

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

Lynch et al. 2013 administered to groups of 10 male and 10 female Harlan Wistar rats an iron trichloride containing complexation/reaction product, termed FemTA by oral gavage. FemTA is a mixture of sodium tartrate [D(–)- and L(+)-tartaric acid and mesotartaric acid], sodium hydroxide, and iron trichloride. The composition of the product was approximately 4% sodium tartrate, 10% mesotartaric acid, 7% chloride, 4% iron, 7% sodium, 0.3% sodium oxalate, and 65% water. FemTA was administered to the groups at dose levels of 500, 1000, and 2000 mg/kg body weight/d (equivalent to 20, 40, or 80 mg of iron/kg body weight/d). Male rats were dosed prior to and during mating and up to the day prior to scheduled sacrifice during the post-mating period (total of 90/91 days).The females were treated with the substance prior to mating and during mating as well as during gestation and lactation (at least up to lactation day 4) (total of 104 to 109 d).. During the treatment period the substance was administered once daily, 7 days per week. A control group was 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, behaviour (functional findings), and gross pathology.

However, compared to the vehicle control, treatment-related effects were observed in rats receiving the substance at dose levels of 1000, and 2000 mg/kg body weight/day. During the haematological examination, an increase in white blood cell count (p < 0.01) and relative neutrophil counts (p < 0.05) as well as a decrease in relative lymphocyte count (p < 0.05) were noted for male rats of the 2000 mg/kg bw/day dose level. Furthermore, an increase in relative eosinophil counts (p < 0.05) were observed in females at the 2000 mg/kg bw/day dose level.

Also, treatment-related effects were observed for clinical biochemical findings. At the 1000 and 2000 mg/kg bw/day dose levels, increased blood urea nitrogen (p < 0.01) and bile acid levels (p < 0.01) were noted for male rats. Furthermore, an increase in alanine aminotransferase (p < 0.01) as well as a decrease in sodium and chloride concentrations were observed in male rats at the 2000 mg/kg bw/day dose level. Increased blood urea nitrogen (p < 0.05) and decreased chloride concentrations (p< 0.05) were recorded for female rats at the 2000 mg/kg bw/day dose level.

At the 500, 1000 and 2000 mg/kg bw/day dose levels, an increased absolute (p < 0.05) and relative kidney weight (p< 0.01) was observed in the male rats. In addition, an increased absolute (p < 0.05) and relative liver weight (p < 0.01) was recorded for male rats of the 2000 mg/kg bw/day dose level. At the 1000 and 2000 mg/kg bw/day dose levels, elevated organ weights were observed for absolute (2000 mg/kg bw/day dose level only; p < 0.01) and relative kidney weights (p < 0.01) for female rats. Alterations in kidney weight were clearly treatment-related, and, given the magnitude, were considered adverse at the 2000 mg/kg body weight/d dose level.

Lastly, treatment-related effects were observed during microscopical examination. Inflammation of the gastro-intestinal tract (increase in both incidence and severity of neutrophilic infiltrates, acute inflammation, goblet / epithelial cell hyperplasia, foci of brown pigment and/or oedema of the rectum, colon and cecum) was observed at the 1000 and 2000 mg/kg bw/day for both sexes and this finding was considered to be treatment-related.

Based on the histopathological findings noted in the gastro-intestinal tract of male and female rats at the 1000 and 2000 mg/kg bw/day dose levels, the no observed adverse effect level (NOAEL) for general toxicity is considered to be 500 mg/kg bw/day for males and females

This reference had several minor reporting and experimental deficiencies:
First, the test substance is mixture composition of sodium tartrate, mesotartaric acid, chloride, iron, sodium, sodium oxalate, and water. Therefore, it is unclear, if the test item-related effects observed in the study are caused by the iron in the mixture or maybe by another compound in the mixture. Furthermore, the low pH of 3.7 alone might cause the inflammation of the gastro-intestinal tract instead of the iron content of the mixture.

According to the guideline the duration of study, following acclimatisation, should be dependent on the female performance and should last approximately 54 days, [at least 14 days pre-mating, (up to) 14 days mating, 22 days gestation, 4 days lactation]. In the study acclimatisation and mating period of the rats were described but clear information of the duration were not provided. In addition, in this study the duration of the premating period was not clearly described. In the method section, it was stated to be 12 weeks. However, according to the provided body weight data it appeared to have been only 11 weeks. Further, in the method section it was described, that male rats were treated for 90/91 days, but the reported body weight data indicate a treatment period of approximately 98 days. A prolongation of the 14 days premating period is beneficial to observe cumulative adverse effects of low magnitude .

The dosage volume was not equal among the treatment groups. In the study, dosing was achieved by the adapting the administrated volume of the test substance. Due to the slight acidic pH (3.7) the irritating aspects of the test substance was in the focus of the study. In the case of irritating substances the guideline foresees no dilution of the test substance.

Pre-treatment data was not provided or mentioned in the study. According to the provided body weight gain data, the rats have been investigated before the treatment.

Furthermore, the authors stated that besides the clinical signs reported, they observed other clinical signs, which were even distributed across treated and control group and were considered to be normal for the age and strain of rat. The type of clinical signs were not further clarified by the authors. Furthermore, it is not clearly stated, if detailed clinical examinations were carried out before treatment was started, as foreseen by the guideline.

Reflecting the more pronounced adverse effects in male rats described above it can be concluded that male rats could be more susceptible to the treatment. The different response of the female mice could be due to the different investigation time points, pregnancy, weaning and lactation. Females were not in a similar state as the males during blood sampling, following pathology and histopathological investigations. Females were sampled at necropsy during the lactation phase. This could explain the different susceptibility of male and female rates in response of the FemTA treatment.

Lastly, historical control data or individual data were not provided. Since the historical control data was not provided, it is not possible to determine, if the findings were within or outside the range of normal biological variation of the rat strain. In addition, individual data would be helpful in order to determine, if the results contain outliners, which might influence the outcome of the results.

Reference 13

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose toxicity study with the reproductive/ developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

Study summary available only. Original reference was not obtainable, but the study was approved by the OECD procedure on Mutual Acceptance of Data (MAD).

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996-03-22

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: Sigma-Aldrich Corporation
- Lot number of test material: 14330TA

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Age at study initiation: 8 weeks
- Weight at study initiation: (P) males: 269.23 – 302.18 g; females: 191.34 – 221.60 g

Route of administration:

oral: gavage

Details on route of administration:

not specified

Vehicle:

not specified

Details on oral exposure:

not specified

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

males: 42 days
females: 42 to 54 days

Frequency of treatment:

daily

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

equivalent to 55 mg Fe/kg bw/day

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

equivalent to 110 mg Fe/kg bw/day

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

equivalent to 220 mg/kg bw/day

No. of animals per sex per dose:

main group: 15 males / 15 females
recovery group: 5 males /5 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: preliminary tests were coducted prior to study conduct. Groups of two male and two female ratsreceived the test substance at dose levels of 60, 125, 250, 500 and 1000 mg/kg bw/day. According to the preliminary tests, all male rats in 1000 mg/kg bw/day treatment group were dead. For female rats, one rat was dead at the same dose level. Therefore, 500 mg/kg bw/day was chosen as the maximum dosage.

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

- Recovery groups: groups were used for the control group and the high dose group (500 mg/kg bw/day)

- Post-exposure recovery period: 2 weeks of post exposure period for recovery groups

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
clinical signs: once a day
mortality: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once a week and right before the necropsy except mating period, but for pregnant females, it was measured on day 0, 7, 14, 20 of gestation period, date of delivery, and 4 days after the delivery.

FOOD CONSUMPTION: Yes
- Time schedule: once a week (except mating period)

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: days 0, 6, 13 and 40 after treatment

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, fasted for 18 hours before necropsy
- How many animals: 5 animals/sex/test group
- Parameters checked: total erythrocyte count (RBC), haemoglobin concentration (HEG), haematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), total leucocyte count (WBC), platelet (PLT), neutrophils (NEU), eosinophils (EOS), basophils (BASO), lymphocytes (LYM), and monocytes (Mono), prothrombin time (PT), activated partial thromboplastin time (APTT) and metheamoglobin (MH)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, fasted for 18 hours before necropsy
- How many animals: 5 animals/sex/test group
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine, total protein, albumin, sodium, potassium, triglycerides, glucose, phosphorus, calcium and cholinesterase. Cholinesterase II actibity was measured with S-butyrylthilcholine iodide as a substrate.

PLASMA/SERUM HORMONES/LIPIDS: Not specified

URINALYSIS: Yes
- Time schedule: not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- How many animals: 5 animals/sex/test group
- Parameters checked: color, specific gravity, pH, glucose, protein, leukocyte and erythrocyte

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before necropsy
- Dose groups that were examined: all test groups
- Battery of functions tested:
1) Sensory organ test: five males and five females were randomly selected from each test group. Both auricle reflex test and corneal reflex test were performed.
2) Motor function test: five males and five females were randomly selected from each test group for traction test.

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A necropsy was carried out in control and treated groups. Furthermore, organ weights of the following organs were obtained for control and treated groups (5 animals/sex/group): testes, epididymider (all males), liver, kidney, adrenal, thymus, spleen, brain and heart

HISTOPATHOLOGY: Yes
Twenty-one tissues were preserved in 10 % buffered neutral formalin solution for histopathologic tests: brain, pituitary, spinal cord, heart, lung, trachea, stomach, ileum, liver, colon, spleen, thyroids, thymus, adrenals, kidneys, urinary bladder, sciatic nerve, bone marrow, uterus, ovaries and lymph node. Testes and epididymides were fixed in bouin’s fixative.

Statistics:

Homogeneity of variance was evaluated using Levene’s test in terms of body weight, food and water consumption, biochemical test of blood and organ weight. When the assumption of homogeneity of variance was met, ANOVA was used. If significant result was observed, Dunnett’s test was used. When the assumption of heterogeneity of variance was met, appropriate data transformation was carried out, then Levene’s test was performed on re-transformed data. If significant result was observed, Dunnett’s test was used.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females
- 25, 250 and 500 mg/kg bw/day: clinical signs such as blackish stool and salivation were observed in treated groups.

- 500 mg/kg bw/day: in the early stages of administration, cases of decrease in locomotion activity were found in both sexes, but these were recovered to normal states. The female rats were more sensitively affected than the male rats in locomotion activity decrease, paleness, emaciation and soiled perineal region. However, these symptoms were reversible within the test period.

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

Mortality:

mortality observed, treatment-related

Description (incidence):

Females:
- 500 mg/kg bw/day: three female rats were found dead on the day 38, 46 and 51. The cause of death was gastrointestinal damages by the test substance.

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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
- 250 and 500 mg/kg bw/day: the rate of body weight gain was significantly decreased in 250 and in 500 mg/kg bw/day male groups (- 4.2 % to - 8.2 % and - 7.5 % to -14 %, respectively) compared to the control group..

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Males and females:
- 500 mg/Kg bw/day: the amount of water consumption was increased for both male and female animals.

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

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females:
- 250 and 500 mg/kg bw/day: both absolute and relative weights of liver were statistically significantly increased in 250 and 500 mg/kg bw/day male groups and in 500 mg/kg bw/day female group. Also, for male rats, absolute adrenal glands weights were statistically significantly increased in 500 mg/kg bw/day group, and relative adrenal glands weights were increased in 250 and in 500 mg/kg bw/day group. Because of hemosiderin deposit in hepatocyte and hyperplasia of zona fasciculate in adrenal cortex, the increased weights of liver and adrenal glands were influenced by the test substance. In 125 mg/kg bw/day male group, liver weight did not differ from the control group, but adrenal glands weights (left) were decreased as compared to the control group.

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females:
- 500 mg/kg bw/day: the following necropsy findings were caused by the test substance: severe diffuse haemorrhagic glandular stomach and severe distension of stomach in dead animals, and diffuse black coloured liver and haemorrhage with diffuse black pigmentation in scheduled necropsy of 500 mg/kg bw/day male group. For females, a case of mass of mesenteric lymph node was observed in 500 mg/kg bw/day group.

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

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males and females:
- 500 mg/kg bw/day: for groups of both sexes, hemosiderin deposit of hepatocyte and glandular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of forestomach, hemosiderin deposit of glandular stomach, neutrophil infiltration of submucosa were observed. These conditions were induced by the test substance and were weaker in females. There were no specific findings in the recovery groups.

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

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

MORTALITY
1) Males
- 0, 125, 250 and 500 mg/kg bw/day: no death was observed for male animals.

2) Females
- 0, 125 and 250 mg/kg bw/day: no death was observed for female animals.

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

BODY WEIGHT AND WEIGHT CHANGES
1) Females:
- 125, 250 and 500 mg/kg bw/day: there was no significant changes except on the day 7 of pre-mating period (decrease of body weight by - 4.9%; 500 mg/kg bw/day dose group), gestation day 7 (decrease of body weight by - 5.1 %; 125 mg/kg bw/day dose group) and the day 4 of lactation period (decrease of body weight by - 7 % and - 6.7 % for 250 and 500 mg/kg bw/day, respectively).

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

FOOD CONSUMPTION
Males and females:
- 0, 125, 250 and 500 mg/kg bw/day: there was no significant difference between the control and the treated groups, and no dose-related change was observed in both sexes.

HAEMATOLOGICAL FINDINGS
Males and females:
Statistically significant differences were found in mean cell volume (MCV), eosinophils (EOS) and platelet (PLT). But these were within the biologically normal range and no dose-dependent changes were evident.

CLINICAL BIOCHEMISTRY FINDINGS
Males and females:
Statistically significant differences were found in cholinesterase (CS), and triglycerides (TG). But these were within the biologically normal range and no dose-dependent changes were evident.

URINALYSIS FINDINGS
Males and females:
- 125, 250 and 500 mg/kg bw/day: there were no specific findings.

BEHAVIOUR (FUNCTIONAL FINDINGS)
Males and females:
- 125, 250 and 500 mg/kg bw/day: both auricle reflex test and corneal reflex test were performed evaluating sensory reflex; no specific reaction was observed
in comparison with the control group. Furthermore, the motor function test showed a significant decrease in females of the 125 and 500 mg/kg bw/day treatment groups. But these decreased values were higher than male control group, since the mean value of female control group was higher than the male control group. There was no significant result in female 250 mg/kg b.w./day group and all male rats. Because there were no dose-dependent changes, motor function was not considered to be affected by iron dichloride.

ORGAN WEIGHT FINDINGS
Females:
- 125, 250 and 500 mg/kg bw/day: for thymus, absolute weight was statistically significantly decreased in female 125 and 500 mg/kg bw/day groups, and relative weight was decreased in 500 mg/kg bw/day group. However, these changes were considered to be individual variations and not due to the test substance.

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

GROSS PATHOLOGICAL FINDINGS
Males and females:
- 0, 125, 250 and 500 mg/kg bw/day: diaphragmatic nodules of liver were sporadically noted in the control and the treated groups. It is a congenital malformation, which is a morphological change and doesn’t have physiological effects.

HISTOPATHOLOGICAL FINDINGS
Males and females:
- 0, 125, 250 and 500 mg/kg bw/day: the following orgnas did not have remarkable results: cerebellum, cerebrum, epididymides, heart, kidney, spleen, thymus and uterus.

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

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

water consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Conclusions:

In the current combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, groups of 15 male and 15 female Sprague Dawley rats were administered via gavage iron dichloride at dose levels of 125, 250 and 500 mg/kg bw/day (equivalent to 55, 110 and 220 mg Fe/kg bw/day). The male and female rats were treated daily for a duration of 42 days and 42 to 54 days, respectively. The treatment period included a two-week pre-mating period as well as mating period, gestation period and lasted up to lactation day 4. A control group without treatment was run concurrently. In addition, recovery groups of five male and five female rats were employed for the control group and the high dose group.

During the observations of the parental male and female rats, no test item-related effects were observed for food consumption, haematology, clinical biochemistry, urinalysis and neurobehaviour. However, clinicals signs such as blackish stool and salivation were observed for both sexes at the 125, 250 and 500 mg/kg bw/day dose levels. Furthermore, in the early stages of administration of 500 mg/kg bw/day, cases of decrease in locomotion activity were found in both sexes, but these were recovered to normal states. The female rats were more sensitively affected than the male rats in locomotion activity decrease, paleness, emaciation and soiled perineal region. However, these symptoms were reversible within the test period. In addition, three female rats of the 500 mg/kg bw/day dose group were found dead on the days 38, 46 and 51. The cause of death was gastrointestinal damages by the test substance. At the 250 and 500 mg/kg bw/day dose levels, the rate of body weight gain of the parental male rats was significantly decreased (- 4.2 % to - 8.2 % and - 7.5 % to -14 %, respectively) compared to the control group and water consumption was increased for male and female parental animals at the 500 mg/kg bw/day dose level compared to the control group.

The investigation of the organ weights of the parental generation revealed that both absolute and relative weights of liver were statistically significantly increased in 250 and 500 mg/kg bw/day male groups and in 500 mg/kg bw/day female group. Also, for male rats, absolute adrenal glands weights were statistically significantly increased in 500 mg/kg bw/day group, and relative adrenal glands weights were increased in 250 and in 500 mg/kg bw/day group. Because of hemosiderin deposit in hepatocyte and hyperplasia of zona fasciculate in adrenal cortex, the increased weights of liver and adrenal glands were influenced by the test substance. In 125 mg/kg bw/day male group, liver weight did not differ from the control group, but adrenal glands weights (left) were decreased as compared to the control group. Furthermore, the following necropsy findings were caused by the test substance: severe diffuse haemorrhagic glandular stomach and severe distension of stomach in dead animals, and diffuse black coloured liver and haemorrhage with diffuse black pigmentation in scheduled necropsy of 500 mg/kg bw/day male group. For females, a case of mass of mesenteric lymph node was observed in 500 mg/kg bw/day group. Lastly, the histopathological examination showed that for groups of both sexes, hemosiderin deposit of hepatocyte and glandular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of forestomach, hemosiderin deposit of glandular stomach, neutrophil infiltration of submucosa were observed at the 500 mg/kg bw/day dose level. These conditions were induced by the test substance and were weaker in females. There were no specific findings in the recovery groups.

Based on the test item-related effects on body weight gain, water consumption, organ weights (liver and adrenal gland), macroscopical findings and microscopical findings, the no observed adverse effect level (NOAEL) for general toxicity is considered to be 125 mg/kg bw/day for males. In addition, the NOAEL for general toxicity is considered to be 250 mg/kg bw/day for females based on mortality, water consumption, organ weight (liver) and microscopical findings.

This reference had several reporting and experimental deficiencies:
Firstly, the test item preparation was not described, as foreseen by the OECD guideline 422. It was not mentioned, if a vehicle was used to administer the test substance to the animals, which makes it impossible to draw any conclusion on the effect of the vehicle on the test system/test substance in case a vehicle was used. Dosing volume of the test substance was also not given. Furthermore, information was missing whether analytical concentration, stability and homogeneity were determined during the study and, therefore, it is unclear, if the animals received the dosages as indicated. Also, as foreseen by the OECD guideline, doses should be given at similar times and adjusted weekly to maintain a constant dose level in terms of animal body weight. There is no indication that this procedure was followed during the study conduct.

The OECD guideline foresees a description of the mating procedure, but information on mating procedure was not fully provided (except for mating period and proof of pregnancy). Furthermore, the authors did not state what happen to the females with no evidence of mating. Information is missing whether these females were further investigated, which could provide further information on the effects caused by the test substance. The authors did not state exactly when the animals were sacrificed during the study. According to the OECD guideline, the study should end on lactation day 4. Looking at some parameters measured it seems that the study ended on lactation day 4, but it not clear stated in description of the study design. Furthermore, recovery groups were used for the control group and the high dose group, as foreseen by the guideline. However, for most occasions the results of the recovery groups were not presented, which makes it impossible to recapitulate, if the effects observed were reversible.

During the study, detailed clinical signs of the parental generation were recorded, however, it is unclear when the observations were also made before treatment with the test item. Due to the missing information, it is not possible to conclude on a change in detailed clinical examination after treatment started. Furthermore, grip strength was not investigated during the neurobehavioural examination of the parental generation, as foreseen by the guideline. Also, clinical chemistry examination was incomplete (total cholesterol, urea and bile acids missing). Histopathology of the parental generation was also not complete. Accessory sex organs and gross lesions, pons, Peyer’s patches and most parts of the intestines were not examined and not all findings of the high dose group were investigated in the low and mid dose groups (e.g. stomach).
According to the OECD guideline, stillbirths, live births and runts should be recorded for the offspring (F1 generation). In addition, live pups should be counted and weighted as well as macroscopical findings of pups that died prematurely should be recorded. This information was not explicitly provided by the authors and no conclusion can be drawn for theses parameters.

Lastly, historical control data or individual data were not provided. Since the historical control data was not provided (except for some reproductive parameters), it is not possible to determine, if the findings were within or outside the range of normal biological variation of the rat strain. In addition, individual data would be helpful in order to determine, if the results contain outliners, which might influence the outcome of the results.

Reference 14

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The following deficiencies can be reported for the publication: test item insufficiently characterised; male rats investigated only; less than three dose levels not investigated; clinical signs recorded, but no indication was made on the time schedule used for recording; observations on mortality missing; incomplete haematology and clinical chemistry; organ weights not determined; histopathology not conducted; individual data missing; historical control data missing

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study, the developmental of a rat model for iron deficiency and toxicological studies was described. Three strains of rats (Fischer-F 344-N, Sprague Dawley, and Wistar/nin) were investigated. Groups of ten animals of each strain received a iron-deficient diet (< 10 ppm; equivalent to < 1.2 mg/kg bw/day) ad libitum for a duration of six weeks. Furthermore, a control group of ten animals of each strain received a diet with an iron content of 220 ppm (equivalent to 26.4 mg/kg bw/day). The following parameters were recorded/examined: clinical signs, body weight gain, food consumption, food efficiency, haematology (haemoglobin concentration, protoporphyrin-to-heme ratio, serum iron concentration), clinical chemistry (liver iron concentration only) and gross pathology.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Specific details on test material used for the study:

not specified

Species:

rat

Strain:

other: Fischer-F 344-N, Sprague Dawley, and Wistar/nin

Details on species / strain selection:

The rat has been used as the model for iron deficiency. It is readily available, and iron deficiency is relatively easy to induce in this species.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Fischer-F 344-N: National Institutes of Health (Bethesda, Md.)
Sprague Dawley: National Institutes of Health (Bethesda, Md.)
Wistar/nin: Wistar Institute (Philadelphia, Pa.)

- Age at study initiation: weanlings
- Housing: housed individually in wire bottomed plastic cages

- Diet (ad libitum):
Stock colony: semisynthetic cereal pulse-based diet
Though the dietary ingredients of this diet differed in some respected from those of the experimental diet of this study, the nutrient composition was essentially the same.

Control and experimental groups: casein, sucrose-starch-based purified diet
The control and experimental diets were identical except that iron was omitted from the diet of the experimental group.
Composition of diet (g/100 g of diet):
Casein: 20
Sucrose: 35
Starch: 35
Groundnut oil: 5
Mineral mix: 4
Vitamin mix: 1
Vitamins A and D and choline chloride were supplemented to required concentrations. Iron as ferrous sulfate was added to the control diet at a concentration of 220 ppm.

Water: deionized water

Regular microbiological monitoring of senitel animals for specific bacteria and virsus indicated that these animals were always maintained in a specific-pathogen-free status.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 °C
- Humidity: 55 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on route of administration:

not specified

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The control and experimental diets were identical except that iron was omitted from the diet of the experimental group.
Composition of diet (g/100 g of diet):
Casein: 20
Sucrose: 35
Starch: 35
Groundnut oil: 5
Mineral mix: 4
Vitamin mix: 1
Vitamins A and D and choline chloride were supplemented to required concentrations. Iron as ferrous sulfate was added to the control diet at a concentration of 220 ppm. The experimental group included <10 ppm iron (iron-deficient group).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

ad libitum

Remarks:

< 10 ppm; equivalent to < 1.2 mg/kg bw/day

Dose / conc.:

220 ppm

Remarks:

equivalent to 26.4 mg/kg/day

No. of animals per sex per dose:

Fischer-F 344-N: 10 male weanlings/group
Sprague Dawley: 10 male weanlings/group
Wistar/nin: 10 male weanlings/group

Details on study design:

not specified

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT GAIN: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption recorded for each animal: Yes, daily

FOOD EFFICIENCY:
- Average body weight gain per feed intake in grams: Yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified
OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: haemoglobin concentration, protoporphyrin-to-heme ratio (P/H), serum iron concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: liver iron concentration

PLASMA/SERUM HORMONES/LIPIDS: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Not specified

The animals were sacrificed by guillotine after the treatment period. The animals were macroscopically examined.

Optional endpoint(s):

not specified

Other examinations:

not specified

Statistics:

Statistical evaluation of the data by Student's t test was initially carried out by comparison of mean values for rats within the same strain fed the control and iron-deficient diets. Subsequently, interstrain comparison between control and iron-deficient diets was done by analysis of variance and multiple t tests. The minimal level of significance was fixed at P = 0.5.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of iron deficiency were evident in the Fischer and Wistar experimental rats. Fur growth was not normal and their eyes were pale.

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of iron-deficient Fischer 344 and Wistar strains (experimental group) was reduced 20 to 30% compared with control rats. The Sprague Dawley iron-deficient rats did not have any difference in body weight compared with their controls.

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of Fischer 344 and Wistar rats fed the iron-deficient diet (experiment group) was decreased 25 to 30% compared with those fed the control diet during the experimental period. This decrease was not evident in the Sprague Dawley iron-deficient rats. These differences in food consumption were reflected in body weight of the three rat strains.

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

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Feed efficiency was reduced to 80% in the Fischer 344 iron-deficient rats and to 65% in the Wistar iron-deficient rats (experiment groups). Feed efficiency of Sprague Dawley iron-deficient (experimental group) and control rats was similar.

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

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Comparison of the mean values for control and iron-deficient groups within each strain indicated that all haematologic variables such as haemoglobin concentration, protoporphyrin-to-heme (P/H), and liver and serum iron concentrations were significantly affected by iron deficiency (experimental groups). However, the magnitude of changes was not the same among various strains. The Fischer strain was the most susceptible to iron deficiency. It had the lowest haemoglobin concentration compared with the other two strains (5.37 versus 6.12 versus 7.42 g/dl, Fischer versus Wistar versus Sprague Dawley respectively; all values were significantly different from each other). Further, serum and liver iron values were lowest and the P/H was highest in Fischer rats compared with the other two strains. The increase in the P/H value confirms development of iron deficiency which, however, was not significant among groups because of small sample size and large variations. Reduction in serum but not liver iron concentration was significantly different among the strains.

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

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathological findings were evident in Fischer and Wistar rats receiving the iron-deficient diet (experimental groups). Internally, the liver was pale, the colon and stomach were distended, and the heart and spleen were enlarged.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

not specified

Remarks on result:

other: see "Remarks"

Remarks:

Clinical signs of iron deficiency were evident in the Fischer and Wistar experimental rats. Fur growth was not normal and their eyes were pale. Furthermore, body weight of iron-deficient Fischer 344 and Wistar strains was reduced 20 to 30% compared with control rats. In addition, food consumption of Fischer 344 and Wistar rats fed the iron-deficient diet was decreased 25 to 30% compared with those fed the control diet. The differences in food consumption were reflected in body weight of the three rat strains. Feed efficiency was reduced to 80% in the Fischer 344 iron-deficient rats and to 65% in the Wistar iron-deficient rats. The Sprague Dawley iron-deficient rats did not have any difference in body weight compared with their controls as well as no decrease in food consumption or feed efficiency compared to controls. Comparison of the mean values for control and iron-deficient groups within each strain indicated that all haematologic variables such as haemoglobin concentration, protoporphyrin-to-heme, and liver and serum iron concentrations were significantly affected by iron deficiency. The Fischer strain was the most susceptible to iron deficiency. Lastly, gross pathological findings were evident in Fischer and Wistar rats receiving the iron-deficient diet. The liver was pale, the colon and stomach were distended, and the heart and spleen were enlarged.

Conclusions:

In this study, the developmental of a rat model for iron deficiency and toxicological studies was decribed. Three strains of rats (Fischer-F 344-N, Sprague Dawley, and Wistar/nin) were investigated. Groups of ten animals of each strain received a iron-deficient diet (< 10 ppm; equivalent to < 1.2 mg/kg bw/day) ad libitum for a duration of six weeks. Furthermore, a control group of ten animals of each strain received a diet with an iron content of 220 ppm (equivalent to 26.4 mg/kg bw/day).

Clinical signs of iron deficiency were evident in the Fischer and Wistar experimental rats. Fur growth was not normal and their eyes were pale. Furthermore, body weight of iron-deficient Fischer 344 and Wistar strains (experimental groups) was reduced 20 to 30% compared with control rats. The Sprague Dawley iron-deficient rats did not have any difference in body weight compared with their controls. In addition, food consumption of Fischer 344 and Wistar rats fed the iron-deficient diet (experiment groups) was decreased 25 to 30% compared with those fed the control diet during the experimental period. This decrease was not evident in the Sprague Dawley iron-deficient rats. These differences in food consumption were reflected in body weight of the three rat strains. Feed efficiency was reduced to 80% in the Fischer 344 iron-deficient rats and to 65% in the Wistar iron-deficient rats (experiment groups). Feed efficiency of Sprague Dawley iron-deficient (experimental group) and control rats was similar.

Comparison of the mean values for control and iron-deficient groups within each strain indicated that all haematologic variables such as haemoglobin concentration, protoporphyrin-to-heme (P/H), and liver and serum iron concentrations were significantly affected by iron deficiency (experimental groups). However, the magnitude of changes was not the same among various strains. The Fischer strain was the most susceptible to iron deficiency. It had the lowest haemoglobin concentration compared with the other two strains (5.37 versus 6.12 versus 7.42 g/dl, Fischer versus Wistar versus Sprague Dawley respectively; all values were significantly different from each other). Further, serum and liver iron values were lowest and the P/H was highest in Fischer rats compared with the other two strains. The increase in the P/H value confirms development of iron deficiency which, however, was not significant among groups because of small sample size and large variations. Reduction in serum but not liver iron concentration was significantly different among the strains.

Lastly, gross pathological findings were evident in Fischer and Wistar rats receiving the iron-deficient diet (experimental groups). Internally, the liver was pale, the colon and stomach were distended, and the heart and spleen were enlarged.

This reference had several reporting and experimental deficiencies:
Firstly, the test item was insufficiently characterised, which makes it impossible to verify the identity of the test item. Furthermore, male rats were only investigated, which makes it impossible to draw any conclusion on the effect iron-deficient diet has on females. In addition, three dose levels were not investigated as foreseen by the OECD guideline. Therefore, a dose-response related analysis cannot be conducted and a No-Observed-Adverse Effect level (NOAEL) cannot be determined. Clinical signs were recorded, but no indication was made on the time schedule used for recording. Furthermore, the authors did not mention, if they recorded/observed any mortality. The table presenting the haematologic parameters does not included all animals (n = 10/group), but there is no explanation why they were excluded. This raises the question, if these animals did not survive until the end of the study. Also, the haematological examination (haemoglobin concentration, protoporphyrin-to-heme ratio and serum iron only) as well as the clinical chemistry examination (liver iron concentration only) were incomplete. In addition, organ weights were not determined, and histopathology of the animals was not conducted. Lastly, individual data and historical control data were missing. Since individual data is missing, it makes it impossible to observe any outliners that might influence the outcome of the study. Due to the fact that the historical control data is missing, it cannot be determined, if the results observed were not within the normal biological variation of the strains investigated and, therefore, a treatment-related effect is observed.

Based on the shortcomings given above, this publication was used as supporting information only.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

2008-09-30

Principles of method if other than guideline:

The 13-week subchronic inhalation study was conducted also according to OECD guidance 39 (2009).

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Rockwood, Torino, Italia

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 2 months
- Weight at study initiation: approx. 228 g (males); 156 g (females)
- Housing: singly housed in polycarbonate cages, containing low‐dust wood shavings as beddingmaterial.
- Diet (ad libitum): standard fixed‐formula diet (KLIBA 3883 pellets maintenance diet for rats and mice (supplier: PROVIMI KLIBA SA, 4303 Kaiseraugst, Switzerland))
- Water (ad libitum): tap water
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40 - 60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.3 µm

Geometric standard deviation (GSD):

1.2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test atmposphere was forced through openings in the inner concentric cylinder of the chamber, towards the rats'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra)
- System of generating particulates/aerosols: WRIGHT DUST FEEDER system (BGI Inc., Waltham, M A, USA)
- Temperature, humidity, pressure in air chamber: controlled and measured continously
- Air flow rate: monitored and controlled continously by calibrated mass flow meters (Hastings HFCC Mass Flow Controllers, Teledyne Hastings-Raydist, Hampton, VA, USA). TYLAN FC-280 S mass flow controller was used for the analytical sampling.
- Method of particle size determination: samples (from breathing zone) analyzed using a BERNERTYPE AERAS low pressure critical orifice cascade impactor. A cyclone was used to prevent particles larger than 10 μm to enter in the inhalation chamber.
- Treatment of exhaust air: purification via aerosol and HEPA filters.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis
- Samples taken from breathing zone: yes, 3 samples/exposure day/ chamber

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations were determined by gravimetric analysis (filter: Glass-fiber-filter, Sartotius, Gottingen, Germany). Filters were evaluated by gravimetric analysis (balance: Mettler AE 100).

Duration of treatment / exposure:

10 animals/sex/group were exposed for 13 consecutive weeks; 10 animal/sex/group were exposed for 13 - 14 consecutive weeks

Frequency of treatment:

6 hours/, 5 days/week

Dose / conc.:

4.7 mg/m³ air (analytical)

Remarks:

± 0.6; 5 mg/m3 (nominal)

Dose / conc.:

16.6 mg/m³ air (analytical)

Remarks:

± 3; 15 mg/m3 (nominal)

Dose / conc.:

52.1 mg/m³ air (analytical)

Remarks:

± 6.4; 50 mg/m3 (nominal)

No. of animals per sex per dose:

20 (10 for haematology, clinical pathology, urinalysis, determination of organ weights and histopathology; 10 for analyses in BAL and iron organ burdens)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: two dose-range finding studies were performed with different Fe oxides (2 weeks) and with Fe3O4 (4 weeks).

** The examined endpoints for the 10 rats/group exposed for 13 weeks were according to the OECD 413, while the remaining 10 rats/group were necropsied 1 -2 weeks later (exposure continued) and were subjected to BAL and analysis of Fe body burden of selected organs.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily; before and after exposure

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly on Mondays and Fridays during the exposure period and once weekly during the post exposure period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: once per week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start and at the end of the exposure period
- Dose groups that were examined: 10 rats/sex/group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13‐week study period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 rats/sex/group
- Parameters examined: erythrocytes (ERY), hematocrit (HCT); hemoglobin (Hb); Hepato Quick (HQUICK); leukocytes (LEUKO); mean corpuscular Hb (MCH); mean corpuscular Hb concentration (MCHC); mean corpuscular volume (MCV); thrombocytes/platelets (THRO).

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the 13‐week study period, uirne was collected over night.
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals: 10 rats/sex/group
- Parameters examined:

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 13–14 week exposure period
- Dose groups that were examined: control and all dose groups
- Number of animals: 10 rats/group/sex
- Parameters examined: TCC, total cell count in BAL; MCD, mean cellular
diameter; MCV, mean cellular volume; LDH, lactate dehydrogenase; AP, alkaline phosphatase; ACPH, acid phosphatase; PROT, protein; PLIPf, phospholipids in BALF; AM, alveolar macrophages; PMN, polymorphonuclear cells; LYM, lymphocytes; EOS, eosinophils; foamy, foamy; NC, cells not classifiable; NAG, β‐N‐acetyl‐glucosaminidase.

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 13 week exposure period
- Dose groups that were examined:
- Number of animals: 10 rats/sex/group
- Parameters examined: Fe3O4 levels in LALN

Sacrifice and pathology:

GROSS PATHOLOGY
clinical pathology was performed at the end of the 13‐week study period using 10 rats per group per sex. Rats were necropsied (surviving rats were sacrificed) and were given a gross-pathological examination. The general physical condition, body orifices, external and internal organs and tissues were examined.

ORGAN WEIGHT:
At the terminal sacrifice, organ weights were determined (adrenal glands, brain, heart, kidneys, liver, lung, lung-associated lymph nodes (LALN), ovaries, spleen, testes, thymus) and organs were persevered for histopathology.

HISTOPATHOLOGY
Examinations were performed to all the organs mentioned above and to a list of tissues. The histopathological evaluations focused on the entire respiratory tract (nasal passages, trachea, lung, lung-associated lymph nodes); it also included all extrapulmonary organs (OECD Guideline 413).

Statistics:

Depending on variates: Dunnet test, Adjusted Welch test, Kruskal-Wallis test followed by Adjusted U tests, Analysis of Variance (ANOVAbctic). A detailed description is provided.

Clinical signs:

effects observed, treatment-related

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:

effects observed, treatment-related

Description (incidence and severity):

- 50 mg/m3: significantly increased neutrophil counts in peripheral blood cytodifferentials occurred in all iron oxide male groups and in female rats.

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 15 and 50 mg/m3: significantly increased lung and LALN weights were observed in both sexes.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 5, 15 and 50 mg/m3: black discolorations of lungs and lung associated lymph nodes were observed macroscopically in almost all Fe3O4 exposure groups, with increasing grading. Black macrophages were seen in the lungs of all exposed groups and they increased in a dose-dependent manner. In all males and females exposed to the upper two concentrations and in the majority of exposed to the lowest concentration, bronchiolar-alveolar hypercellularity and black macrophages in BAL were detected.
- 15 and 50 mg/m3: examinations revealed focal pigmented macrophages in the trachea of many substance-exposed rats. The paracortical area of LALNs appeared stastistically significantly enlarged in all the males and almost all females exposed to the the upper two concentrations.
- 50 mg/m3: in the nasal cavity, eosinophilic epithelial globules occured in both males and females exposed at the highest two concentrations. Epithelial metaplasia was observed in some of the male rats and focal inflammatory infiltrates occured in some females. Serius-red staining of the lung resulted in the observation of increased collagenous fibers in all animals of both sexes at the highest concentration.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID:
- 5, 15 and 50 mg/m3: alveolar macrophages appeared significantly increased already at the dose of 5 mg/m3. A concentration-dependent increase was observed in polymorphonuclear cells (PMNs) at 5 mg/m3 and above. Slight increases of phospholipids in BAL fluid were detected in animals exposed to the highest concentration, while BAL protein levels increased already at 5 mg/m3, albeit slightly.
- 15 and 50 mg/m3: A remarkable increase of total cell count (TCC) occured in animals exposed to the upper two concentrations. The LDH measurements revealed signs of cytotoxicity at 15 mg/m3 and above. Beta-N-acetyl-glucosaminidase activity appeared elevated in both sexes; this was the case for acid phosphatase in female rats.
The results of the BAL analysis are summarized in Table 11 (see attachment).

LUNG BURDEN:
- 5, 15 and 50 mg/m3: iron oxide translocation was minimally increased above the background iron levels of this organ at low-dose (approx. 30 times below the LALN iron‐burden observed at mid-dose). However, the respective increase at mid- and high-dose was proportional to the exposure concentration. Lung iron levels were determined but could not be interpreted due to a protocol error (iron analysis in lavaged lungs).

Details on results:

MORTALITY:
- The exposures were tolerated without mortality.

BODY WEIGHT AND WEIGHT CHANGES:
- The exposures were tolerated without concentration‐dependent significant changes in body weights.

FOOD CONSUMPTION:
The exposures were tolerated without concentration‐dependent significant changes in food consumption.

WATER CONSUMPTION:
The exposures were tolerated without concentration‐dependent significant changes water consumption.

OPHTHALMOLOGICAL FINDINGS:
- No abnormality in ophthalmology (performed prior to the start and towards the end of study) was observed.

HAEMATOLOGICAL FINDINGS:
- 5 and 15 mg/m3: PMN counts were still within the range of historical control data.

URINALIS FINDINGS:
- Urinalysis did not demonstrate differences across groups (data not shown).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- Absolute and relative (vs body weights) organ weights were confounded by the inconsistently higher body weight gains of the male control rats. Toxicologically conclusive changes in the remaining organ weights were not observed when using the organ‐to‐brain weight ratios.

GROSS PATHOLOGICAL FINDINGS:
- General clinical pathology, including hemostasis did not demonstrate differences across groups (data not shown).

HISTOPATHOLOGICAL FINDINGS:
- Adverse extrapulmonary effects of Fe3O4 were not observed at any concentration.
- 5, 15 and 50 mg/m3: diffuse tubular atrophy/degeneration was detected in 1 high-dose rat and in 3 rats from each of the other concentration groups. As stated by the authors, corresponding to these lesions, spermatic debris occured in the epididymides. These findings are not related to the concentration of the substance and in several animals they occured unilaterally. Therefore, they are not exposure related.
- Testes: (Multi)-focal tubular atrophy and degeneration was seen in all groups including the controls.

Dose descriptor:

NOAEC

Effect level:

4.7 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

immunology

organ weights and organ / body weight ratios

Critical effects observed:

not specified

LUNG BURDEN - HALF-TIME:

The time- and particle volume-dependence of lung burdens of Fe3O4 in rats exposed for 13 weeks for 6 h/day on 5 day/week in the present study were modeled. The following half-time for each concentrations were calculated: 5 mg/m3: 94 days; 15 mg/m3: 177 days; 50 mg/m3: 392 days. According to the athor, the lung overload treshold was reached after inhalation from a concentration of 15 mg/m3. The exposure concentrations selected for the 13 week study were modeled to attain the overload threshold (NOAEL) at 5 mg/m3 with overload‐dependent pulmonary inflammation at 15 and 50 mg/m3. The overload‐dependent delay in clearance was expected to be in the range of t1/2≈1 year at 50 mg/m3.

 

BRONCHOALVEOLAR LAVAGE FLUID:

Many cells in the BAL fluid could not be clearly differentiated due to extreme loading and were classified as 'non-classifiable cells'. However, most non-classifiable cells were assumed to be alveolar macrophages by the authors of the study report.

Conclusions:

In the subchronic inhalation toxicity study by Pauluhn (2006a), iron(II,III) oxide (Fe3O4) aerosols were administered to Wistar rats (20 male and 20 female per group) by the dynamic directed-flow nose-only technique; actual mean concentrations were 0, 4.7 ±0.6, 16.6 ±3 and 52.1 ±6.4 mg/m3 air; the animals were exposed for 6 h/day, 5 days/week over a period of 13 weeks. Ten rats/group were necropsied 1 - 2 weeks later (exposure continued). Particles had a MMAD of 1.3 µm and GSD ~2.
In a subchronic inhalation study, 20 male and female Wistar rats were exposed to aerosolized Fe3O4 at concentrations of 4.7, 16.6 and 52.1 mg/m3 (Pauluhn (2006a); Pauluhn (2011)) by the dynamic directed-flow nose-only technique. The animals were exposed for 6 h/day, 5 days/week over a period of 13 weeks. Ten rats/group were necropsied 1 - 2 weeks later (exposure continued). Particles had a MMAD of 1.3 µm and GSD ~2. Clinical signs were recorded daily before and after exposure. Body weights were recorded twice weekly, and food and water consumption were determined once a week. Also, an ophthalmology was performed prior to start and the end of the exposure period. At sacrifice, inflammatory endpoints were determined in bronchoalveolar lavage (BAL). Rats were subjected to gross pathological examination and histopathology (adrenal glands, brain, heart, kidneys, liver, lung, LALNs, ovaries, spleen, testes, thymus).
The exposure was not associated with any specific clinical signs and consistent changes in body weights. Haematology, clinical pathology and urinalysis were unobtrusive.
The NOAEC was 4.7 mg/m3, based on the findings from BAL analysis (increased PMNs, protein levels) and histopathology. Mild and borderline changes were considered to be associated with the exposure to poorly soluble particles rather than specific toxicity of the tested particles. The effects found at higher concentrations appear to be consistent with a particle-overload related inflammatory response.

The results of this inhalation RDT study in Wistar rats can generally be regarded as reliable without restrictions, since the study was conducted according to OECD guideline 412 (2008) and under GLP.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-11-08 to 2006-02-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only males were tested in the study. There were no haematological or clinical biochemistry analyses performed. Adrenals were not weighed or histopathologically examined after sacrifice. Humidity of air in the chamber during the exposure was very low. No description of the method for determining the iron content in the organs.

Reason / purpose for cross-reference:

other: Reference to dose range finding study

Reason / purpose for cross-reference:

other: Reference to dose range finding study

Reason / purpose for cross-reference:

other: Reference to dose range finding study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only male animals were tested; no haematology or clinical biochemistry; noorgan weights and histopathology of adrenals; verly low humidity in chamber air during exposure; postexposure period of 6 months.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Weight at study initiation: approx. 229.6 g
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type III or IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.5 µm

Geometric standard deviation (GSD):

2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 180 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 22.1 - 24.0 °C and 0.27 - 1.63 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- Method of particle size determination: samples (from breathing zone) analyzed using a BERNERTYPE AERAS low pressure critical orifice cascade impactor. A cyclone was used to prevent particles larger than 10 μm to enter in the inhalation chamber.
- Treatment of exhaust air: purification via aerosol (dust) and HEPA filters.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric analysis
- Samples taken from breathing zone: yes, 3 samples/exposure day/chamber

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations were determined by gravimetric analysis (filter: Glass-fiber-filter, Sartotius, Gottingen, Germany). Filters were evaluated by gravimetric analysis (balance: Mettler AE 100).

Results:
Test atmosphere: analytical monitoring of the aerosol test atmospheres from the breathing zone (filter samples) as well as the continuous realtime aerosol monitorin indicated that the exposure conditins were temporally stable during the 6-h exposure periods and that they were reproducible during the course of study.
Concentrations: analytical concentrations (10.1 ± 1.4, 19.7 ± 3.3, 45.6 ± 6.8 and 95.8 ± 17.6 mg/m3) met the targeted concentrations of 10, 20, 50 and 100 mg/m3.

Duration of treatment / exposure:

4 consecutive weeks

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

10.1 mg/m³ air (analytical)

Remarks:

±1.44; 10 mg/m³ (nominal)

Dose / conc.:

19.7 mg/m³ air (analytical)

Remarks:

±3.27; 20 mg/m³ (nominal)

Dose / conc.:

45.6 mg/m³ air (analytical)

Remarks:

±6.77; 50 mg/m³ (nominal)

Dose / conc.:

95.8 mg/m³ air (analytical)

Remarks:

±17.59; 100 mg/m³ (nominal)

No. of animals per sex per dose:

10 males in main study (four dose groups and control group); 20 animals for recovery study (four dose groups and control group)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: based on a preliminary dose range finding study by Pauluhn (2005). To assess the concentration-response and time course of pulmonary clearance impairment and the persistence of inflammation (if any) utilized also published evidence from preveious inhalation exposures to TiO2 (Warheit et al. (1997)).*
In the dose range finding study by Pauluhn, Wistar rats (48 male rats per group) were exposed by inhalation (dynamic directed flow nose-only) to aerosolized iron oxide powder (Ferroxide Black 88P (Magnetite)) for 6 hrs/day on 5 days/week for two consecutive weeks (days 0-11) at a mean actual concentration (i.e., breathing zone concentrations) of 185.6 mg/m3 air. DQ12 was served as reference dust (positive control for lung damage) in a concentration of 200.4 mg/m3. Throughout the groups, the aerosol was highly respirable to rats (MMAD: < 2 μm; GSD: < 3). During the 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, 107. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory endpoints were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. The repeated exposure to the aerosolized Magnetite was not associated with any specific clinical signs, changes in body temperatures or body weights. Changes in LOH, phospholipids, alkaline phosphatase, protein, P-NAG, and collagen were observed in the iron oxide group at all time points. BAL-cell count and BAL-cell volume in the exposed group reached the level of the concurrent air control group on day 58. DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the postexposure period. Splenic iron was examined on days 15 and 29, but not at day 58 and 107, due to a lack of any consistent effect and the high variability in iron content of this organ. A remarkable increase of iron was observed in lungs and in LALN with evidence of time-related translocation from one compartment (lung) to another (LALN). At the end of the postexposure period, a significant increase of iron was observed in LALN. The iron content determined in testes and liver were indistinguishable amongst the groups. Based on the iron determination made in lung tissue over a postexposure period of three months, the elimination half-time was 208 days. There were no toxicologically significant changes in absolute or relative organ weights except the elevated absolute and relative weights of lungs and LALNs. DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the postexposure period. At the end of the exposure period intraalveolar granular material was seen in all rats exposed to iron-oxide. These granules and alveolar macrophages appeared to be black. At the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals exposed to iron oxide. At all interim sacrifices iron oxide particles were detected in BALT, but not in liver, kidneys, and testis. These findings could not be detected in liver, kidneys or testis at the end of the exposure period and after 2 weeks of recovery. Soluble iron staining (Prussian Blue staining) of the lung (alveolar region) was positive at all time points in the exposed group. However, this positive iron staining was evident within/around alveolar macrophages, whilst the interstitium did not show evidence of solubilized, i.e., potentially bioavailable iron. Prussian Blue staining of hepatic tissue was unobtrusive.
In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm). Particle retention half-time was approximately 330 days for 250 mg/m3. The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. Due to this dose-response relationship for TiO2 dust in this study and in order to achieve a meaningful investigation of the concentration-response relationship, the target concentrations of 10, 20, 50 and 100 mg / m3 were selected.
- Rationale for the study design: based on a comparative 2-week inhalation study (Pauluhn, 2005)
- Post-exposure recovery period in satellite groups: approx. 6 months (1, 8 and 24 weeks)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalation of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded daily before and after exposure or once per week during the postexposure period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week (on fridays and mondays) during the study and once per week (on monday) during the exposure and postexposure periods

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 4-week exposure period and during the course of a 6 months postexposure period subgroups of rats were serially sacrificed following 1, 8, and 24 weeks after exposure and examined.
- Dose groups that were examined: control and all treatment groups from the main and recovery study
- Number of animals: 5 rats/group/serial sacrifice
- Parameters examined: total number of lavaged cells (BALC), including the volume and diameter; Lactate dehydrogenase (LDH), total protein, alkaline and acid phosphatase, and β-N-Acetyl-glucosaminidase (β-NAG).

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 4-week exposure period and at each serial sacrifice during the course of a 6 months postexposure period.
- Dose groups that were examined: control group and all treatment groups from the main and recovery study
- Number of animals: 5 rats/group/serial sacrifice
- Parameters examined: iron coontent in lungs (right lobes), lung-associated lymph nodes (LALN), and liver.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated. The necropsy was a systematic gross examination of each animals`s general physical condition, body orifices, external and internal organs and tissues.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: brain, heart, kidneys, liver, lungs, lung-associated lymph nodes, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
For the histopathological examination, the following organ tissues were fixed and examined: head (with nasal cavity), kidneys, liver, lungs (left lobe), spleen, testes, thymus, and trachea. Organs and tissues or representative pieces of them were fixed in 10 % neutral buffered formalin of Davidson`s solution respectively. The lungs were instilled by the intratracheal route (20 cm water coloumn) with 10 % neutral buffered formalin and then postfixed with the other organs in 10 % buffered formalin. The lungs were embedded in Paraplast and sections were prepared from the lungs. All slides were steined with haematoxylin and eosin. Additionally, a Prussian-Blue staining and a Sirius-Red staining were carried out. However, they were not evaluated because of the absence of progressive changes or lung remodeling.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and Kruskal-Wallis test followed by adjusted U test.

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):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 50 and 100 mg/m3: lung and LALN weights were significantly increased with evidence of a time-related diminution of changes. The elevation in lung weights decreased in intensity during the course of the 6 months postexposure period and borderline to significant effects still existed at 50 and 100 mg/m3.
- 100 mg/m3: the increased brain weight did not show any consistent concentration-dependence.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

During necropsy, gray or black discolouration/s was detected in lungs and lung-associated lymph nodes (LALNs) of all substance-exposed rats at the first sacrifice time-point after one week of recovery. After 8 and 24 weeks of recovery, the disolouration of the lungs and LALNs was still seen. Enlargement of LALNs was observed in rats at 50 mg/m3 and above. After 24 weeks, a gray or black discolouration of LALNs was detected in most of the rats from all iron oxide exposure groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 10, 20, 50 and 100 mg/m3: at the 1 week after the end of exposure sacrifice, black and/or enlarged intraalveolar macrophages with phagocytized particles were seen in all substance-exposed ratsrevealing a clear shift in grading from minimal findings (grade 1) at the low concentration to a more moderate categorization (grade 3) in the high-level exposure group. Macrophages appeared to be arranged in clusters distributed all over the tissue of the lung lobes. Black intraalveolar granules occured in a concentration-dependent manner, providing clear evidence the external exposure concentrations matched the lung dose. Similarly, hypercellularity of the bronchiolar-alveolar region, predominantly adjacent to macrophage clusters, was detected. In the bronchus-associated-lymphoid (BALT), black macrophages and/or black pigment was seeen in all exposure groups. Following a postexposure period of 8 and 24 weeks, the findings were not distinctly different from those observed after one week of recovery, although, black intraalveolar granules occured only in rats from the highest concentration.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE:
results from cytodifferentiation are considered to be uninterpretable.
- 20, 50 and 100 mg/m3: in the acellular supernatant increased protein and LDH were observed. These elevations decreased in intensity during the course of the 6 months postexposure period and borderline to significant effects still existed at 50 and 100 mg/m3.

IRON CONTENT IN TISSUES:
- 10, 20, 50 and 100 mg/m3: a statistically significant increase of iron content was observed in the lung tissue all treatment groups (p < 0.01 and p < 0.05) in week 36, 85 and 197.
- 20, 50 and 100 mg/m3: there was a significant increase in iron content of lung-associated lymph nodes (LALN) in the highest dose group in week 36 (p <0.01). A statistically significant increase of iron content was also observed in the LALN of the 20, 50 and 100 mg/m3 groups in week 85 and 197 (p < 0.01 and 0.05). However, iron content in lung tissue decreased within the respective treatement group from week 36 to 197. Assuming a single-compartment 1st order elimination kinetics from the lung, the elimination half-times for Magnetite were 53, 79, 109 and 165 days in rats exposed to 10, 20, 50 and 100 mg/m3, respectively.

Details on results:

CLINICAL SIGNS:
- all rats tolerated the exposure without specific signs.
- 100 mg/m3: rat no. 140 showed during the postexposure period (days 49 - 84) red nasal encrustations.

MORTALITY:
- the exposure was tolerated without mortality and morbidity.

BODY WEIGHT AND WEIGHT CHANGES:
- there was no statistically significant difference in body weights amongst the groups.
- There was no statisticially significant changes in the body weight gain during the 4-week exposure period.
- 10, 20, 50 and 100 mg/m3: statistically significant changes in the body weight gain were observed in all treatment groups in weeks 25 - 28 and 28 - 35 (p < 0.01 and 0.05). A significant decrease was observed in the 10 and 100 mg/m3 groups in week 56 - 63 (p < 0.01), and in the 10 mg/m3 group in week 168 - 175. The body weight gain in the 10 mg/m3 group was significantly increased in week 63 - 70.

BRONCHOALVEOLAR LAVAGE:
- evidence of sustained lysosomal processes did not exist at any time points.
Cytodifferentiation was complicated due to the extreme dust loading of BAL-cells. This resulted in the following shortcomings: (I) many cells could not be appropriately differentiately differentiated due to their black colour leading to an increase in `non-classifiable cells (NC)`; (II) the increased loading of cells with density poorly soluble particles resulted in technical problems during cytocentrfugation with associated inhomogeneities of the cell density in cytospots.

IRON CONTENT IN TISSUES:
- 50 mg/m3: significant increase in the iron content of liver tissue was observed in week 197 (p < 0.01).

Dose descriptor:

NOAEC

Effect level:

10.1 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

In the publication of Pauluhn (2011), the time- and particle volume-dependence of lung burdens of Fe3O4 in rats exposed for 4 weeks for 6 h/day on 5 day/week in the present study were modeled. The following half-time for each concentrations were calculated: 10 mg/m3: 89 days; 20 mg/m3: 116 days; 50 mg/m3: 174 days; 100 mg/m3: 282 days. The overload threshold is defined as 5 mg/m3 particle volume(alveolar fraction) according to Pauluhn (2011). The 4‐week study was designed so that this threshold was not attained at 10mg/m3 and minimally exceeded at 20mg/m3 at the end of the exposure period. At 50 and 100 mg/m3, pulmonary toxicity was expected to occur without complete reversibility at the end of the 6 month post exposure period.

 

According to the athor, the lung overload treshold was reached after inhalation from a concentration of 20 mg/m3.*

 

*Reference:

- Pauluhn, J. (2011): Subchronic inhalation toxicity of iron oxide (magnetite, Fe3O4) in rats: pulmonary toxicity is determined by the particle kinetics typical of
poorly soluble particles. J. Appl. Toxicol. 2012; 32: 488–504.

Conclusions:

In this RDT study, aerosolized Magnetite powder were administered by inhalation at concentrations of 10, 20, 50 and 100 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow nose-only for 6-hours/day on 5 days/week for 4 consecutive weeks. During a 6 month postxposure period subgroups of rats (ten per sacrifice) were serially sacrificed 1, 8, and 24 weeks after the 4 week exposure period and examined.
According to the author, the evaluation of the lungs of rats exposed to four different concentrations of Magnetite revealed findings clearly consistent with a 'poorly soluble particle' effect after the 4-week exposure period followed by a 6-month postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extrapulmonary organs to any appreciable extent was not observed. Extrapulmonary effects causally linked to the exposure of Magnetite were not found at any exposure concentration and time point. At the end of the 6-month postexposure period, the findings causally linked to the exposure to iron oxide (e.g. bronchiolo-alveolar hypercellularity, septal thickening) showed a decrease in incidence and/or severity and occurred essentially only at 95.8 mg/m3, a concentration shown to be high enough to cause a self-sustained type of inflammation due to a substantially delayed clearance of particles (t1/2 = 165 days) as a result of lung overload. Thus, the results of this study support the view that the NOAEC (no-observed adverse effect concentration) of Magnetite is 10.1 mg/m3 with borderline effects at 19.7 mg/m3, a concentration considered to be at the transition of lung overloading (delayed clearance of alveolar macrophages due to the increased engulfment of insoluble particles).

The results of this inhalation RDT study in Wistar rats can generally be regarded as reliable with restrictions, since the study was conducted according to OECD guideline 412 (1998) and under GLP.
However, only males were tested in the study. There were no haematological or clinical biochemistry analyses performed. In addition, the adrenals were not weighed or histopathologically examined after sacrifice. Furthermore, the humidity of air in the chamber during the exposure was very low. There is also no description of the method for determining the iron content in the organs.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Publication shows significant methodological deficiencies in the experimental set up and documentation. Test material was insufficiently characterised (no purity or impurities). Only male rats were tested. The type of inhalation exposure were not specified. The method for verification of carbonyl iron concentrations in the exposure air was not described.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male rats were exposed to carbonyl iron particles 6 hours/day, 5 days/week for 4 weeks via inhalation at concentrations of 5, 50, and 250 mg/m³ and evaluated at selected intervals through 6 months postexposure (0 hour, 1 week, and 1, 3, and 6 months). Indices of pulmonary inflammation as well as alveolar macrophage clearance functions (morphology, in vivo and in vitro phagocytosis, and chemotaxis), cell proliferation, and histopathology endpoints were measured at several post-exposure time periods through 6 months. In addition, amounts of carbonyl iron in lungs and tracheobronchial lymph nodes were measured to allow an evaluation of particle clearance and translocation patterns.

GLP compliance:

not specified

Remarks:

in publication.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: GAF Corp., New York
- mean diameter of particles: 0.25 µm, but generally forms 1.0-µm agglomerates.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: particles were heated to 200 °C for 4 hr to eliminate the possibility of endotoxin contamination.

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 7- 8 weeks old

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.9 - <= 3.4 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating aerosols: methods utilized for aerosol generation of carbonyl iron particles have previously been reported (Warheit et al. (1991)).*
According to Warheit et al. (1991), the atmosphere was generated with a K-tron bin feeder equipped with twin feed screws. The dust was metered into a polycarbonate transfer tube where high pressures of air swept the test material into the exposure chamber. Chamber concentration was maintained by controlling the dust-feed rate into the generation apparatus, or by varying the air-flow rate.*

*References:
- Warheit, D. B., Carakostas, M. C., Hartsky,M. A., and Hansen, J. F. (1991):
Development of a short-term inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107, 350–368.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Overall mean atmospheric concentrations were very close to the design concentrations.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

4.8 mg/m³ air (analytical)

Remarks:

± 2 mg/m3; nominal concentration: 5 mg/m3

Dose / conc.:

51.8 mg/m³ air (analytical)

Remarks:

± 15 mg/m3; noinal concentration: 50 mg/m3

Dose / conc.:

243.6 mg/m³ air (analytical)

Remarks:

± 90 mg/m3; nominal concentration: 250 mg/m3

No. of animals per sex per dose:

10 males per group, divided into 5 animals for each particle concentration - for lung tissue studies (lung burden, lung clearance, pulmonary cell proliferation, time course of particle translocation to tracheobronchial lymph nodes, and lung histopathology) and 5 rats for each group for bronchoalveolar lavage studies - cell differentials/lung inflammatory responses, alveolar functional responses- chemotaxis, in vitro phagocytosis.**

**Annotation: this information(s) given is not included in the publication but was obtained through personal correspondence with the author Warheit, D.B.

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure recovery period in satellite groups: 1 week, and 1, 3 and 6 months

Positive control:

Not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs and mortality): animals were cheched daily by the DuPont Haskell Animal Resources group.**

DETAILED CLINICAL OBSERVATIONS: not specified
BODY WEIGHT: not specified
FOOD CONSUMPTION: not specified
WATER CONSUMPTION: not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

BRONCHOALVEOLAR LAVAGE FLUID (BALF): yes
Bronchoalveolar lavage procedures and biochemical assays on lavaged fluids were conducted according to methods previously described (Warheit et al. (1991))*.
- Time schedule for analysis: after 4 weeks of exposure, and after 3 and 6 months of recovery period.
- Dose groups that were examined: control group and all dose groups
- Parameters examined:
(I) Phagocytosis: Alveolar macrophage cell culture and phagocytosis assay methods have previously been reported (Warheit et al. (1984a,b)).* For the phagocytic assay for carbonyl iron-exposed macrophages, a suspension of latex particles was similarly opsonized with serum. The latex particles ranged from 2 to 4 mm in diameter. A similar mass concentration of latex particles (i.e., 1.75 mg/ml) was added to the monolayers.
(II) Chemotaxis: alveolar macrophages were collected from carbonyl iron- or sham-expmed rats by lavage as described above. The chemotaxis assay was carried out as described previously using three concentrations (1, 5, and 10%) of zymosan-activated sera as the chemotactic stimulus (Warheit er al., 1984b, 1992)*
(III) Pulmonary cell proliferation studies: pulmonary cell proliferation experiments were conducted according to methods previously described (Warheit et al. (1992))*.

LUNG BURDEN: Yes
- Time schedule for analysis: after 4 weeks of exposure
- Dose groups that were examined: control group and all dose groups
- Parameters examined: analyses of lung and lymph node burdens were conducted by digesting tissue specimens in hydrofluoric acid and analyzing for iron, using the method of ICP-AES.

**Annotation: this information(s) given is not included in the publication but was obtained through personal correspondence with the author Warheit, D.B.

*References:
- Warheit, D. B., Carakostas, M. C., Hartsky, M.A., and Hansen, J. F. (1991): Development of a shortterm inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica. Toxicol. Appl. Pharmacol. 107, 350-368.
- Warheit, D. B., Hill, L. H ., and Brody, A. R. (1984a): Surface morphology and correlated phagocytic capacity of pulmonary macrophages lavaged from the lungs of rats. Exp. Lung Res. 6, 71-82.
- Warheit, D.B., and Chang, L.Y., Hill, L.H., Hook, G.E.R., Crapo, J.D., and Brody, A.R. (1984b): Pulmonary macrophage accumulation and asbestos-induced lesions at sites of fiber deposition. Am. Rev. Respir. Dis. 129, 301-310.
- Warheit, D.B., Kellar, K.A., and Hartsky, M.A. (1992): Pulmonary cellular effects in rats following aerosol exposures to ultrafine Kevlar aramid fibrils: Evidence for biodegradability of inhaled fibrils. Toxicol. Appl. Pharmacol. 116, 225-239.

Sacrifice and pathology:

GROSS PATHOLOGY: No data

HISTOPATHOLOGY: Yes
The lungs of rats exposed to carbonyl iron particles for 4 weeks were prepared for light microscopy by airway infusion using methods previously reported (Warheit et al. (1984b, 1991)). Histopathology were performed after 4 weeks of exposure, and after 1 week, 1 and 6 months of postexposure period.

Statistics:

Statistics were carried out using a two-tailed Student t test on a Microsoft Excel software program (p<0.05).

Clinical signs:

no effects observed

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

5, 50 and 250 mg/m3:
- lesions in the respiratory system varied with exposure concentration and duration of post-exposure recovery.
- 5 mg/m³ produced only minimal effects. Particle-laden macrophages and a minimal diffuse increase in alveolar macrophages (histiocytosis) were evident at 0 days of recovery. The histiocytosis was no longer evident at 1 week postexposure. Individual particle-laden macrophages could be found in very low numbers within air spaces and lymphoid tissue throughout the entire 6-month postexposure period.
- 50 and 250 mg/m³ produced a wide spectrum of effects within the lung. Free granular pigment of CI was present on the mucosal surfaces of bronchioles and bronchi at 0 days of recovery. Particle-laden macrophages, found individually, were numerous throughout the air spaces at this same time period. Beginning at 1 week post-exposure and persisting thereafter, many dense aggregates of particle-laden macrophages were within alveoli and alveolar ducts. Cellular hypertrophy and hyperplasia were evident at alveolar wall and duct bifurcations that were adjacent to macrophage aggregates. Mucosal hypertrophy and hyperplasia were also observed within the bronchi and bronchioles. Number of pigment-laden macrophages found individually and in aggregates was much greater in animals exposed to the highest concentration, and occupied a greater portion of the lung. Severity of cellular hypertrophy and hyperplasia at alveoli and alveolar duct bifurcations was significantly greater in animals exposed to the highest dose.
- Severity and character of the lesions changed with time. Free granular pigment was no longer apparent at 1 week post-exposure in any concentration group. Particle-laden macrophages, found individually, decreased in number with time, but were evident in small numbers within the pulmonary air spaces throughout the entire 6-month recovery period. The numbers and size of the dense aggregates of macrophages within alveoli and alveolar ducts, increased during the first month post-exposure but did not expand throughout the remaining 5-month post-exposure period. Minimal mucosal hypertrophy and hyperplasia in bronchi and bronchioles were evident at 1 month postexposure in the mid- and high-dose groups. Focal cellular hypertrophy and hyperplasia were associated with aggregates of pigmented macrophages, and were evident at alveoli and alveolar duct bifurcations for the entire 6-month postexposure period in the mid- and high-dose groups. Pigmented macrophages could also be observed within pulmonary lymphoid tissue throughout this time period.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID:
- 50 mg/m3: rats demonstrated small but sustained inflammatory responses which reached control levels by 1 month postexposure. LDH and protein values in BAL fluids recovered in exposed rats.
- 250 mg/m3: Four-week exposures to inhaled carbonyl iron particles produced sustained pulmonary inflammatory responses in animals. Nearly 100% of alveolar macrophages recovered by lavage from rats carbonyl iron contained particles 6 months after the termination of exposures. Animals had significant pulmonary inflammation 3 months following a 4-week exposure period. LDH and protein values in BAL fluids were elevated in rats (data not shown).

LUNG BURDEN:
- 250 mg/m3: carbonyl iron particles produced significant exposure-related accumulations of particles in the lungs of exposed rats. Rats had lung burdens of nearly 2000 mg/g of fixed lung tissue or 17 mg/lung.

PARTICLE TRANSLOCATION TO TRACHEOBRONCHIAL LYMPH NODES:
- 250 mg/m3: inhaled carbonyl iron particles translocated to tracheobronchial lymph nodes.

Details on results:

CLINICAL SIGNS:
- no reported (major) effects were reported by this group.**

BRONCHOALVEOLAR LAVAGE FLUID:
- No significant increases relative to controls in BAL alkaline phosphatase and N-acetylglucosaminidase values were measured for any exposure groups (data not presented).

**Annotation: this information(s) given is not included in the publication but was obtained through personal correspondence with the author Warheit, D.B.

Dose descriptor:

NOAEC

Effect level:

> 4.8 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

Male rats were exposed to carbonyl iron particles 6 hours/day, 5 days/week for 4 weeks via inhalation at concentrations of 5, 50, and 250 mg/m³ and evaluated at selected intervals through 6 months postexposure (0 hour, 1 week, and 1, 3, and 6 months). Indices of pulmonary inflammation as well as alveolar macrophage clearance functions (morphology, in vivo and in vitro phagocytosis, and chemotaxis), cell proliferation, and histopathology endpoints were measured at several post-exposure time periods through 6 months. In addition, amounts of carbonyl iron in lungs and tracheobronchial lymph nodes were measured to allow an evaluation of particle clearance and translocation patterns.
According to the authors, exposure to high dust concentrations of carbonyl iron particles produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition.

According to the authors, lesions in the respiratory system varied with exposure concentration and duration of postexposure recovery. Only minimal effects were reported from the lowest dust concentration (4.8 mg/m³). In addition, total granulocyte (PMN) cell counts in the bronchoalveolar fluid (BALF) of the rats from the low-dose group (< 1*10^5) were comparable to that of the shame controls at all study time points. The concentration of 51.9 mg/m³ carbonyl iron displayed slight to moderate increased total cell numbers of granulocytes in the BALF (> 1*10^5 - 2*10^6 at day 180) post exposure. At the concentration of 243.6 mg/m³, the total granulocyte count in the BALF were increased by more than 2*10^6 during the 180 days of post exposure. Therefore, a NOAEC of 4.8 mg/m³ can be concluded which is supported by unaffected alveolar macrophage function, unaffected proliferation index (BrdU labelling) of proximal lung parenchymal cells and comparable proliferation index of terminal bronchiolar cells.

Deficiencies:
The publication shows significant methodological deficiencies in the experimental set up and documentation. Test material was insufficiently characterised (no purity or impurities). Only male rats were tested. The type of inhalation exposure was not specified. The method for verification of carbonyl iron concentrations in the exposure air was not described.

Reference 4

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The purity of the test material was not specified. This study was not in accordance with any subacute inhalation toxicity guideline and only male rats were used for the toxicity evaluation. The number of animals per group and time point (n = 6) was low, and thus, robustness is only limited. A justification for the concentration tested is not provided. The use of only one dose group precludes dose-response relationship evaluations. Details on the test animals (e.g. weight at study initiation and test group randomisation) and housing conditions (e.g. environmental conditions and group sizes) are missing. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development, clinical chemistry, haematology, and histopathology results of other organs than lung is missing. Methodology on lung burden analysis and aerosol generation is insufficiently described.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Warheit, D.B. et al. (1991) investigated on the pulmonary response in male rats (Crl:CD BR) after short-term inhalation to carbonyl iron aerosol. Groups of 6 or 14 rats were exposed to carbonyl iron aerosol at a concentration of 110 mg/m³ (analytical) or air for 6 h per day over a total period of 3 days via nose-only inhalation. The bronchoalveolar lavage fluid (BALF) was analysed for total cell count, differential cell count, total protein level, alkaline phosphatase activity, and lactate dehydrogenase activity directly after the last exposure as well as 1 day, 2 days, 8 days, 1 month, and 2 months after the last exposure. Moreover, the rat lungs were examined via histopathology and TEM analysis. Further, airway and parenchymal cell turnover was determined in a radio-labelling experiment. The lung burden was estimated based on data of a parallel acute study.

GLP compliance:

not specified

Remarks:

in publication

Limit test:

no

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was heated to 250°C for 4 hr to eliminate possible endotoxin contamination.

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, NY, USA)
- Age at study initiation: 8 weeks

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.6 µm

Geometric standard deviation (GSD):

1.7

Remarks on MMAD:

97% of the particles were determined to be <10 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Cylindrical polycarbonate or stainless-steel holders equipped with conical nose pieces
- Method of holding animals in test chamber: restrainers
- System of generating particulates/aerosols: The atmosphere was generated with a K-tron bin feeder equipped with twin feed screws. The dust was metered into a polycarbonate transfer tube where high pressures of air swept the test material into the exposure chamber. Chamber concentration was maintained by controlling the dust-feed rate into the generation apparatus, or by varying the air-flow rate.
- Method of particle size determination: Particle size measurements of airborne particles in the test chamber were determined with a Sierra cascade impactor.

TEST ATMOSPHERE
- Brief description of analytical method used: For gravimetric analysis, samples of atmospheric carbonyl iron were taken at approximately 30-min intervals by drawing calibrated volumes of chamber atmosphere through pre-weighed glass-fiber filters.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see above ("Details on inhalation exposure")

Duration of treatment / exposure:

3 days

Frequency of treatment:

6 hours/day

Dose / conc.:

110 mg/m³ air (analytical)

Remarks:

± 42 mg/m³

No. of animals per sex per dose:

30 males (test material exposed; 6 per time point for analysis) or 98 males (sham control; 14 per time point for analysis)

Control animals:

yes, sham-exposed

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: not specified
DETAILED CLINICAL OBSERVATIONS: not specified
BODY WEIGHT: not specified
FOOD CONSUMPTION: not specified
WATER CONSUMPTION: not specified
OPHTHALMOSCOPIC EXAMINATION: not specified
HAEMATOLOGY: not specified
CLINICAL CHEMISTRY: not specified
URINALYSIS: not specified
NEUROBEHAVIOURAL EXAMINATION: not specified
IMMUNOLOGY: not specified

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 0 hrs, 24 hrs, 48 hrs, 8 days, 1 month, and 2 months after last exposure
- Dose groups that were examined: treated and sham control group
- Number of animals: 6 (treated group) or 14 (sham control group) rats per time point
- Parameters checked: total cell count, macrophage number, granulocyte number, lactate dehydrogenase, alkaline phosphatase, protein level

LUNG BURDEN: Yes (estimates of retained dose were made for rats exposed to carbonyl iron for 3 days. Estimates from an acute study performed in parallel.)
- Time schedule for analysis: after the 6 h exposure
- Dose groups that were examined: treated group
- Number of animals: not specified
- Parameters checked: retained iron dose determined via ICP-MS

Sacrifice and pathology:

GROSS PATHOLOGY: No data

HISTOPATHOLOGY: Yes
- The lungs of carbonyl iron-treated, or sham control rats were prepared for light or electron microscopy by vascular or airway perfusion at recovery periods of 0, 24, and 48 hr; 8 days; and/or 1, 2, or 3 months following aerosol exposure. Sagittal sections of the left lung were made with a razor blade. Tissue blocks were dissected from upper, middle, and lower regions of the lung, and were subsequently prepared for light microscopy (paraffin embedded, sectioned, and haematoxylin-eosin stained), as well as for transmission electron microscopy (post-fixed, dehydrated, uranyl acetate/lead-citrate stained ultrathin sections).

Other examinations:

LUNG CELL PROLIFERATION:
- Airway and lung parenchymal cell turnover: 2 groups each of carbonyl iron-exposed rats and corresponding sham controls were pulsed 48 hr after a 3-day exposure. An intraperitoneal injection of 2 µCi/g body wt of tritiated [³H]thymidine was given 1 hr prior to termination. Lungs were fixed, removed, and sectioned (parasagittal sections from the cranial, middle, and caudal regions of the left lobes) and mounted on microscope slides. were dehydrated and embedded in glycol methacrylate. Afterwards, slides were coated with type NTB2 emulsion, developed, and stained with haematoxylin and eosin. A minimum of 2000 cells each were counted in terminal bronchiolar and alveolar regions. For each treatment group, labelled nuclei (i.e., five or more grains on the nucleus) in airways (i.e., terminal bronchiolar epithelial cells) or lung parenchyma (i.e., epithelial cells, interstitial cells, and macrophages) were counted using light microscopy.

Statistics:

A one-way analysis of variance (ANOVA) and Bartlett’s test were calculated for each sampling time. When the F test from ANOVA was significant, the Dunnett test was used to compare means from the control group and the group exposed to carbonyl iron. Significance was judged at the 5% probability level. The data were subsequently normalized and are represented as a percentage of sham control values for that experiment.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LUNG BURDEN:
- Estimate for retained iron was 650 µg/lung.

Details on results:

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
- Carbonyl iron (CI) exposure did not alter the total numbers of cells recovered by lung lavage. The viability of cells recovered in CI-exposed rats was greater than 96% and was not significantly different from sham controls.
- Cell differential analyses of carbonyl iron exposed rats demonstrated that the numbers of macrophages remained constant following the 3-day exposure and at every time period postexposure.
- No significant differences in protein concentration and enzyme activities (i.e. LDH and ALP) were observed at any time postexposure following carbonyl iron exposure.

LUNG CELL PROLIFERATION:
- No increases in the labelling index were measured in the lungs of carbonyl iron-exposed rats.

HISTOPATHOLOGY:
- No pulmonary lesions were detected at any time postexposure in rats exposed to carbonyl iron particles for 3 days.

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Conclusions:

Warheit, D.B. et al. (1991) investigated on the pulmonary response in male rats (Crl:CD BR) after short-term inhalation to carbonyl iron aerosol. Groups of 6 or 14 rats were exposed to carbonyl iron aerosol at a concentration of 110 mg/m³ (analytical) or air for 6 h per day over a total period of 3 days via nose-only inhalation. The bronchoalveolar lavage fluid (BALF) was analysed for total cell count, differential cell count, total protein level, alkaline phosphatase activity, and lactate dehydrogenase activity directly after the last exposure as well as 1 day, 2 days, 8 days, 1 month, and 2 months after the last exposure. Moreover, the rat lungs were examined via histopathology and TEM analysis. Further, airway and parenchymal cell turnover was determined in a radio-labelling experiment. The lung burden was estimated based on data of a parallel acute study.

The lung burden in carbonyl iron-exposed rats was estimated to be 620 µg iron/lung. Short-term inhalation exposure did not induce significantly altered BALF parameters at any time point, when compared to the sham control group. The airway and parenchymal cell turnover rates were not significantly different from controls. The histopathological examination did not reveal any pulmonary lesions at any time post exposure.

Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information.

The purity of the test material was not specified. This study was not in accordance with any subacute inhalation toxicity guideline and only male rats were used for the toxicity evaluation. The number of animals per group and time point (n = 6) was low, and thus, robustness is only limited. A justification for the concentration tested is not provided. The use of only one dose group precludes dose-response relationship evaluations. Details on the test animals (e.g. weight at study initiation and test group randomisation) and housing conditions (e.g. environmental conditions and group sizes) are missing. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development, clinical chemistry, haematology, and histopathology results of other organs than lung is missing. Methodology on lung burden analysis and aerosol generation is insufficiently described.

The study was considered not reliable [RL=3], due to significant methodological deficiencies.

Reference 5

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-04-30 to 2004-08-11

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

It is a dose range finding study conducted according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only one dose and only males were tested; no haematology and clinical biochemistry in blood; organ weight of adrenals were missing; histopathology of adrenals and heart were missing.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Mean body weight at study initiation: 227.54 mg
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of an impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.43 µm

Geometric standard deviation (GSD):

2.14

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHTDUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 200 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 22.7 °C and 14.3 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- Method of particle size determination: samples (from breathing zone) analyzed using a BERNERTYPE AERAS low pressure critical orifice cascade impactor. A cyclone was used to prevent particles larger than 10 μm to enter in the inhalation chamber.
- Treatment of exhaust air: purification via cotton-wool and HEPA filters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations were determined by gravimetric analysis (filter: Glass-fiber-filter, Sartotius, Gottingen, Germany). Filters were evaluated by gravimetric analysis (balance: Mettler AE 100).

Duration of treatment / exposure:

2 consecutive weeks

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

210.2 mg/m³ air (analytical)

Remarks:

± 46.7 mg/m3

Dose / conc.:

200 mg/m³ air (nominal)

No. of animals per sex per dose:

48 males per group: 12 males per group in main study (one dose group, air control and positive control group); 12 males/group/serial sacrifice in recovery study (one dose group, air control and recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm).* Particle retention half-time was approximately 330 days for 250 mg/m3. The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. For this 2-week inhalation study the dosimetrically adjusted concentrations are 104 and 504 mg/m³ air. From the dose-response relationship of the Warheit et al. (1997) study it can be appreciated that a meaningful comparison can only be achieved near the inversion point of the slopes of the respective dose- response curves rather than in the ranges of the amplitudes of maximum responses.* Based on these considerations twice the intermediate concentration of the titanium dioxide study was considered most appropriate. Therefore, the initial target concentration of this 2-week inhalation study was 250 mg/m³. During the pre-trials and at the commencement of study it was technically difficult to generate this concentration on a day-to-day basis. Therefore, in order to minimize fluctuations in concentrations, all target concentrations were adjusted to approx. 200 mg/m³.
- Post-exposure recovery period in satellite groups: up to 3 months (4, 18, 57 and 96 days)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalation of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

DQ12 (Quartz; 200.4 mg/m3) for lung damage

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded at least daily before and after exposure and once per week during the exposure-free days.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were measured before exposure, on a twice per week basis on Fridays and Mondays, and after the exposure-free weekends (Mondays).

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58 and 107 and examined.
- Dose groups that were examined: control, positive control and treatment group from the main and recovery studies.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: recovery of lavage fluid, total cell count in BAL, mean cellular diameter (MCD), mean cellular volume (MCV), lactate dehydrogenase (LDH), alkaline phosphatase, collagen, acid phosphatase, total protein, phospholipids in BALF, β-N-Acetyl-glucosaminidase (β-NAG) in BALF.

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, and 107.
- Dose groups that were examined: control group and treatment group from the main and recovery studies.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: iron content in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organ tissues were fixed and examined: trachea, lung (left lobe), kidneys, spleen, liver, testes (left), thymus, and all organs of tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. The lungs were instilled by the intratracheal route with 10 % neutral buffered formalin (20 cm water column) and then postfixed with the other organs in 10 % neutral buffered formalin. All slides were stained with haematoxylin and eosin (H&E) and with Prussian Blue.

Other examinations:

RECTAL TEMPERATURE:
Rectal measurements was made 5 rats/groups on days 0, 4, and 11. shortly after cessation of exposure using a digital thermometer equipped with a rectal probe for rats.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and
Kruskal-Wallis test followed by adjusted U test.

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):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Main study: absolute and relative lung weights, and relative lung-associated lymph nodes (LALN) weights were statistically significant increased compared to the air control group (p < 0.01). Relative kidney weights were significantly decreased compared to air controls (p < 0.01).

- Recovery study: relative organ weights of LALN were statistically significantly increased at all time points of the postexposure period compared to air controls (p < 0.01 and p < 0.05).

- Positive control (DQ12): absolute and relative organ weights of lung and LALN were statistically significantly increased in the main study and at all time points during postexposure period compared to the control group (p <0.01).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Exposed group: red discolouration of lungs and enlarged lung associated lymph nodes (LALN) were observed. Discolouration of the LALN was seen in all rats from this group at the end of exposure and during the whole recovery period (1 - 3 recovery period).
- Positive control (DQ12): grey discolouration of lungs was observed. All lungs from this group during all recovery necropsies were less collapsed upon opening of the thoracic cavity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposed group: at the end of the exposure period intraalveolar granular material was seen in all rats exposed to iron-oxide. These granules as well as the alveolar macrophages appeared to be red. At the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals. At all interim sacrifices iron oxide particles were detected in BALT.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID
Exposed groups:
- At all time points the average recovery of the lavage fluid instilled into the lung was high, i.e., it exceeded 80 % of the instilled volume.
- Main study: statistically significant increases in were observed in total cell count in BAL (TCC), mean cellular volume (MCV), lactate dehydrogenase (LDH), total protein (PROT) and phospholipids in BALF (PLIPF) compared to the air control group (p < 0.01 and p < 0.05).

- Recovery study: also, TCC were statistically significant increased in all recovery periods compared to air control group. There were statistically significant increases in mean cellular diameter (MCD) and MCV in the first recovery period, and in the levels of LDH and PROT in the second and third recovery period compared to air controls (p < 0.01 and p < 0.05).

- Positive control: DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the course of the postexposure period. In the main study, statistically significant changes were observed in total cell count
in BAL, LDH, AP, ACPH, total protein, and β-NAG compared to the control group (p < 0.01 and p < 0.05). These parameters, as well as the collagen and phospholipids in BALF were significantly increased at all time points during the postexposure period compared to the control group (p < 0.01 and p < 0.05).
For more information, please refer to table 1 in the field "attached background material" field below.

IRON DETERMINATION IN ORGAN TISSUES:
- Exposed group: a remarkable increase of iron was observed in lungs and in LALN with evidence of time related translocation from one compartment to another. The iron content in these organs were statistically significant increased at all time points in the postexposure period compared to the control group (p < 0.01 and p < 0.05).

IRON KINETICS IN LUNG AND LALN:
Assuming a single-compartment 1st order elimination kinetics from the lung, the elimination half-time for the test item was 87 days.

Details on results:

CLINICAL SIGNS:
air control and exposed group: all rats tolerated the exposure without specific signs. As far as clinical signs were observed in the exposed group they were related to incidental and isolated findings without any time-related exacerbation.

MORTALITY:
No mortality occurred.

BODY WEIGHT AND BODY WEIGHT GAIN:
there was no statistically significant difference in body weights amongst the groups.
Exposed groups: statistically significant changes in body weight gain were observed on days 0 – 4, 21 – 35, 63 - 70 and 77 – 84 compared to the air control group (p < 0.01 and p < 0.05).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
no significant changes were observed in the absolute and relative organ weights of brain, heart, thymus, liver, and spleen in the main study and at all time points during postexposure period compared to the control group. In the 2nd time point of postexposure period, the relative testes weights were statistically different compared to the control group (p < 0.05).

GROSS PATHOLOGICALFINDINGS: ???

HISTOPATHOLOGICAL FINDINGS:
At all interim sacrifices in the examined extrapulmonary organs (liver, kidneys, and testis) were no evidence of translocated iron particles existed. No iron oxide related findings could be detected in the liver, kidneys and testis at the end of the exposure period and after 2 weeks of recovery.

BALF MEASUREMENTS:
Lipidperoxidation (TBARS) was measured in BALC and lung tissue and no distinct differences amongst the group was observed. Cytodifferentiation was planned however, omitted due to the extreme dust loading of BALC.

IRON DETERMINATION IN ORGAN TISSUES:
- Exposed group: during the recovery study (at day 58), statistically significant increase was observed in the testes (p < 0.05) compared to air control group. There were no significant changes in the iron content of the liver at all time points in the postexposure period compared to air control group. Splenic iron was examined on days 15 and 29. Due to a lack of any consistent effect and in the light of the spontaneously occurring variability in iron content, iron determinations in spleen were omitted on days 58 and 107.

RECTAL TEMPERATURE:
Exposed group: in comparison to the concurrent air control group, there was no evidence of a conclusive, significant effect on body (rectal) temperature in the iron oxide group, whilst positive control (DQ12) elicited a hypothermic response on days 0 and 4.

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Conclusions:

In this RDT study, aerosolized hematite was administered by inhalation at a concentration of 210.2 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow noseonly for 6-hours/day on 5 days/week for 2 consecutive weeks. During a 3-month postexposure period, subgroups of rats (12 per sacrifice) were serially sacrificed 15, 29, 58, and 107 days and examined. DQ12 was served as reference dust (positive control for lung damage; MMAD (GSD): 2.27 µm (1.84)) in a concentration of 200.4 mg/m3. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory end points were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. aerosol was highly respirable to rats (MMAD: 1.43 μm; GSD: 2.14).
According to the author, 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 postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extra-pulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxide was not detected at any time point. At the end of the 3-month postexposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity) showed a decrease in incidence and/or severity.

This study was conducted as dose range finding study according to OECD 412 (1981) and under GLP.
However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

Reference 6

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-04-30 to 2004-08-11

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

It is a dose range finding study conducted according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only one dose and only males were tested; no haematology and clinical biochemistry in blood; organ weight of adrenals were missing; histopathology of adrenals and heart were missing.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Mean body weight at study initiation: 228.84 mg
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of an impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry air

Mass median aerodynamic diameter (MMAD):

1.52 µm

Geometric standard deviation (GSD):

1.95

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHTDUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 200 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 22.0 °C and 17.3 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- Method of particle size determination: samples (from breathing zone) analyzed using a BERNERTYPE AERAS low pressure critical orifice cascade impactor. A cyclone was used to prevent particles larger than 10 μm to enter in the inhalation chamber.
- Treatment of exhaust air: purification via cotton-wool and HEPA filters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations were determined by gravimetric analysis (filter: Glass-fiber-filter, Sartotius, Gottingen, Germany). Filters were evaluated by gravimetric analysis (balance: Mettler AE 100).

Duration of treatment / exposure:

2 consecutive weeks

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

200 mg/m³ air (nominal)

Dose / conc.:

185.6 mg/m³ air (analytical)

Remarks:

± 31.4 mg/m3

No. of animals per sex per dose:

48 males per group: 12 males per group in main study (one dose group, air control and positive control group); 12 males/group/serial sacrifice in recovery study (one dose group, air control and recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm).* Particle retention half-time was approximately 330 days for 250 mg/m3. The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. For this 2-week inhalation study the dosimetrically adjusted concentrations are 104 and 504 mg/m³ air. From the dose-response relationship of the Warheit et al. (1997) study it can be appreciated that a meaningful comparison can only be achieved near the inversion point of the slopes of the respective dose- response curves rather than in the ranges of the amplitudes of maximum responses.* Based on these considerations twice the intermediate concentration of the titanium dioxide study was considered most appropriate. Therefore, the initial target concentration of this 2-week inhalation study was 250 mg/m³. During the pre-trials and at the commencement of study it was technically difficult to generate this concentration on a day-to-day basis. Therefore, in order to minimize fluctuations in concentrations, all target concentrations were adjusted to approx. 200 mg/m³.
- Post-exposure recovery period in satellite groups: up to 3 months (4, 18, 57 and 96 days)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalati on of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

DQ12 (Quartz; 200.4 mg/m3) for lung damage

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded at least daily before and after exposure and once per week during the exposure-free days.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were measured before exposure, on a twice per week basis on Fridays and Mondays, and after the exposure-free weekends (Mondays).

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58 and 107 and examined.
- Dose groups that were examined: control, positive control and treatment group from the main and recovery studies.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: recovery of lavage fluid, total cell count in BAL, mean cellular diameter, mean cellular volume, lactate dehydrogenase (LDH), alkaline phosphatase, collagen, acid phosphatase, total protein, phospholipids in BALF, β-N-Acetyl-glucosaminidase (β-NAG) in BALF.

LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, and 107.
- Dose groups that were examined: control group and treatment group from the main and recovery study.
- Number of animals: 6 rats/group/serial sacrifice
- Parameters examined: iron content in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organ tissues were fixed and examined: trachea, lung (left lobe), kidneys, spleen, liver, testes (left), thymus, and all organs of tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. The lungs were instilled by the intratracheal route with 10 % neutral buffered formalin (20 cm water column) and then postfixed with the other organs in 10 % neutral buffered formalin. All slides were stained with haematoxylin and eosin (H&E) and with Prussian Blue.

Other examinations:

RECTAL TEMPERATURE:
Rectal measurements was made 5 rats/groups on days 0, 4, and 11. shortly after cessation of exposure using a digital thermometer equipped with a rectal probe for rats.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and
Kruskal-Wallis test followed by adjusted U test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 200 mg/m3: motility reduced, limp (rat no. 138 which succumbed on day 55). This clinical signs were related to incidental and isolated findings without any time-related exacerbation.
- Positive control (DQ12): tachypnea, irregular breathing patterns, labored breathing patterns, hair-coat ungroomed. Signs occurred in a biphasic manner.

Mortality:

mortality observed, treatment-related

Description (incidence):

- 200 mg/m3: 1 single rat (no. 138) succumbed on day 55. Although a clear causal relationship cannot be established.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 200 mg/m3: absolute organ weights of lung and lung-associated lymph nodes (LALN) were only statistically significantly increased in the main study, and their relative organ weights were increased at all time points during postexposure period compared to the control group (p <0.01 and p < 0.05).
- Positive control (DQ12): absolute and relative organ weights of lung and LALN were statistically significantly increased in the main study and at all time points during postexposure period compared to the control group (p <0.01).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Exposed group: black discolouration of lungs and enlarged lung associated lymph nodes (LALN) were observed. Discolouration of the LALN was seen in all rats from this group at the end of exposure and during the whole recovery period (1 - 3 recovery period).
- Positive control (DQ12): grey discolouration of lungs was observed. All lungs from this group during all recovery necropsies were less collapsed upon opening of the thoracic cavity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 200 mg/m3: at the end of the exposure period intraalveolar granular material was seen in all rats exposed to iron-oxide. These granules as well as the alveolar macrophages appeared to be black. At the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals. At all interim sacrifices iron oxide particles were detected in BALT.
Soluble iron staining (Prussian Blue) of the lung (alveolar region) was positive at all time points and this positive staining was evident within/around alveolar macrophages.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID:
- At all time points the average recovery of the lavage fluid instilled into the lung was high, i.e., it exceeded 80% of the instilled volume.
- 200 mg/m3: in the main study, statistically significant increases were observed in total cell count in BAL, LDH, acid phosphatase, total protein, and β-N-Acetyl-glucosaminidase (β-NAG) compared to the control group (p < 0.01 and p < 0.05). At all time points of the postexposure period, significant increases were observed in total cell count in BAL, mean cellular volume, LDH, alkaline phosphatase (AP), acid phosphatase (ACPH), and total protein compared to controls (p < 0.01 and p < 0.05).
- Positive control: DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the course of the postexposure period. In the main study, statistically significant changes were observed in total cell count in BAL, LDH, AP, ACPH, total protein, and β-NAG compared to the control group (p < 0.01 and p < 0.05). These parameters, as well as the collagen and phospholipids in BALF were significantly increased at all time points during the postexposure period compared to the control group (p < 0.01 and p < 0.05).
For more information, please refer to table 1 in the field "attached background material" field below.

IRON DETERMINATION IN ORGAN TISSUES:
- 200 mg/m3: a remarkable increase of iron was observed in lungs and in LALN with evidence of time-related translocation from one compartment to another. The iron content in these organs were statistically significantly increased at all time points in the postexposure period compared to the control group (p < 0.01).

Details on results:

CLINICAL SIGNS:
- Control: all rats tolerated the exposure without specific signs.

BODY WEIGHT AND WEIGHT CHANGES:
- there was no statistically significant difference in body weights among the groups.
- 200 mg/m3 and positive control: statistical significances, especially to body weight gais, are considered to be of no toxicological relevance.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
- 200 mg/m3: no significant changes were observed in the absolute and relative organ weights of brain, heart, thymus, liver, spleen, and kidneys in the main study and at all time points during postexposure period compared to the control group. The absolute and relative organ weights of testes were not significantly different compared to the controls in the main or postexposure period. In the 1st time point of postexposure period, the relative testes weights were statistically different compared to the control group (p < 0.05).

HISTOPATHOLOGICAL FINDINGS:
No iron oxide related findings could be detected in the liver, kidneys and testis at the end of the exposure period and after 2 weeks of recovery. At all interim sacrifices, in the examined extrapulmonary organs (liver, kidneys, and testis) existed no evidence of translocated iron particles. During the Iron staining with Prussian Blue, the interstitium did not show evidence of solubilized, i.e., potentially bioavailable iron. Prussian Blue staining of hepatic tissue was unobtrusive.

BRONCHOALVEOLAR LAVAGE FLUID:
- Lipidperoxidation measured in BALC and lung tissue showed no distinct differences amongst the groups.
- Cytodifferentiation was planned, however, omitted due to the extreme dust loading of BALC.

RECTAL TEMPERATURE MEASUREMENT:
In comparison to the concurrent air control group, there was no evidence of a conclusive, significant effect on body (rectal) temperature in the iron oxide group, whilst DQ12 (positiv control group) elicited a hyperthermic response on days 0 and 4.

IRON DETERMINATION IN ORGAN TISSUES:
- 200 mg/m3: during the recovery study, statisticially significant increase were observed in the liver tissue at day 29 and in the testes at day 58 (p < 0.05). At the other time points, no significant changes were observed in these two organs. Splenic iron was examined on days 15 and 29. Due to a lack of any consistent effect and in the light of the spontaneously occurring variability in iron content, iron determinations in spleen were omitted on days 58 and 107.

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Conclusions:

In this RDT study, aerosolized magnetite powder was administered by inhalation at a concentration of 185.6 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow noseonly for 6-hours/day on 5 days/week for 2 consecutive weeks. During a 3-month postexposure period, subgroups of rats (12 per sacrifice) were serially sacrificed 15, 29, 58, and 107 days and examined. DQ12 was served as reference dust (positive control for lung damage; MMAD (GSD): 2.2.7 µm (1.84)) in a concentration of 200.4 mg/m3. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory end points were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. aerosol was highly respirable to rats (MMAD: 1.52 µm; GSD: 1.95).
According to the author, 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 postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extra-pulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxide was not detected at any time point. At the end of the 3-month postexposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity) showed a decrease in incidence and/or severity.

This study was conducted as a dose range finding study according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

Reference 7

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-04-30 to 2004-08-11

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

It is a dose range finding study conducted according to OECD 412 (1981) and under GLP. However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981-05-12

Deviations:

yes

Remarks:

only one dose; only males tested; no haematology and clinical biochemistry; no organ weight of adrenals; no histopathological examination of adrenals and heart.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd Cpb:WU (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 2 - 3 months
- Mean body weight at study initiation: 227.61 mg
- Housing: during the study periods the animals were housed singly in conventional Makrolon Type IIIh cages; bedding material: type BK 8/15 low-dust wood granulate, supplier: Ssniff, Soest/Westfalen, Germany.
- Diet (ad libitum): standard fixed-formula diet (KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice), supplier: PROVIMI KLIBA SA, 4303 Kaiseraugust, Switzerland.
- Water (ad libitum): tap water
- Acclimation period: approx. 1 week

DETAILS OF FOOD AND WATER QUALITY:
Available data provided no evidence of an impact on the study objective. Results of food and water analyses are retained by Bayer HealthCare AG.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 40 - 60 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (approx. 14 watt/m2 floor area)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

conditioned dry

Mass median aerodynamic diameter (MMAD):

1.31 µm

Geometric standard deviation (GSD):

2.16

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: test atmospheres were generated using a WRIGHT DUST FEEDER system (BGI Inc., Waltham, MA 02154, USA).
- Inhalation chamber: inner diameter: 14 cm; outer diamer: 35 cm (two-chamber system); height 25 cm; internal volume: 3.8 L.
- System of generating particulates/aerosols: test atmospheres were generated using a WRIGHTDUST FEEDER system (BGI Inc., Waltham, MA, USA). For dry powder dispersion, conditioned compressed dry air (30 L/min.; generic dispersion pressure: approx. 200 kPa) was used. Test item was metered in a reservoir and then was compressed to a pellet. From this pellet defined amounts of test item were scraped off and entrained into the main air flow. The airborne powder was then conveyed into the inner cylinder of the inhalation chamber. Then, after humidification, the test atmosphere was forced through openings in the inner concentric cylinder of the chamber, directly towards the rats 'breathing zone (direct-flow). The stability of the test atmosphere was monitored continously using an aerosol real-time device (vide infra).
- Temperature, humidity, pressure in air chamber: controlled and measured continously; 21.7 °C and 21 % rel. humidity.
- Air flow rate: 45 L/min; inlet air flow: 30 L/min.
- Air change rate: 158 air changes per hour (45 L/min. x 60 min./(3 x 3.8 L)), continuous generation of test atmosphere.
- Method of particle size determination: samples (from breathing zone) analyzed using a BERNERTYPE AERAS low pressure critical orifice cascade impactor. A cyclone was used to prevent particles larger than 10 μm to enter in the inhalation chamber.
- Treatment of exhaust air: purification via cotton-wool and HEPA filters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations were determined by gravimetric analysis (filter: Glass-fiber-filter, Sartotius, Gottingen, Germany). Filters were evaluated by gravimetric analysis (balance: Mettler AE 100).

Duration of treatment / exposure:

2 consecutive weeks

Frequency of treatment:

6 hr/day, 5 days/week

Dose / conc.:

200 mg/m³ air (nominal)

Dose / conc.:

195.7 mg/m³ air (analytical)

Remarks:

± 19.3 mg/m3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the inhalation toxicity study by Warheit et al. (1997), male rats were exposed to TiO2 6 hr/day, 5 days/week, for 4 weeks at actual concentrations of 51.9 and 252 mg/m3 (MMADs: 1.4 - 1.7 μm).* Particle retention half-time was approximately 330 days for 250 mg/m3.
The impact of this TiO2 dust load and similar lung burdens produced a sustained pulmonary inflammatory response measured through a period of 3-6 months postexposure concomitant with increases cell labeling of terminal airway and pulmonary parenchymal cells. The results of this study demonstrate that exposure to high dust concentration of this innocuous particle type produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. With regard to dose-response 51.9 mg/m3 caused minimal toxicity whereas exposure to 252 mg/m3 caused a precipitous increase in the inflammatory endpoints. For this 2-week inhalation study the dosimetrically adjusted concentrations are 104 and 504 mg/m³ air. From the dose-response relationship of the Warheit et al. (1997) study it can be appreciated that a meaningful comparison can only be achieved near the inversion point of the slopes of the respective dose- response curves rather than in the ranges of the amplitudes of maximum responses.* Based on these considerations twice the intermediate concentration of the titanium dioxide study was considered most appropriate.
Therefore, the initial target concentration of this 2-week inhalation study was 250 mg/m³. During the pre-trials and at the commencement of study it was technically difficult to generate this concentration on a day-to-day basis. Therefore, in order to minimize fluctuations in concentrations, all target concentrations were adjusted to approx. 200 mg/m³.
- Post-exposure recovery period in satellite groups: up to 3 months (4, 18, 57 and 96 days)

*Reference:
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M.A. (1997). Inhalation of High Concentrations of Low Toxicity Dusts in Rats Results in Impaired Pulmonary Clearance Mechanisms and Persistent Inflammation. Toxicol. Appl. Pharmacol. 145, 10-22.

Positive control:

DQ12 (200.4 mg/m3) for lung damage

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded at least daily before and after exposure and once per week during the exposure-free days.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were measured before exposure, on a twice per week basis on Fridays and Mondays, and after the exposure-free weekends (Mondays).

FOOD CONSUMPTION AND COMPOUND INTAKE: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58 and 107 and examined.
- Dose groups that were examined: control, positive control and treatment group from the main and recovery studies.
- Number of animals:
- Parameters examined: recovery of lavage fluid, total cell count in BAL, mean cellular diameter, mean cellular volume, lactate dehydrogenase (LDH), alkaline phosphatase, collagen, acid phosphatase, total protein, phospholipids in BALF, β-N-Acetyl-glucosaminidase (β-NAG) in BALF.
LUNG BURDEN: Yes
- Time schedule for analysis: at the end of the 2-week exposure period and during the course of a 3 months postexposure period subgroups of rats were serially sacrificed on days 15, 29, 58, and 107.
- Dose groups that were examined: control group and treatment group from the main and recovery studies.
- Number of animals:
- Parameters examined: iron content in lungs, lung-associated lymph nodes (LALN), spleen, liver, and testes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving rats were sacrificed at the end of the respective observation period using sodium pentobarbital as anaesthetic and complete exsanguination by severing of the abdominal aorta. All rats, irrespective of the day of death, were given a gross pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.

ORGAN WEIGHTS:
The following exsanguinated organs were weighed: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes and thymus. The organ-to-body relationships are specified in both absolute and relative terms.

HISTOPATHOLOGY: Yes
The following organ tissues were fixed and examined: trachea, lung (left lobe), kidneys, spleen, liver, testes (left), thymus, and all organs of tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes were also fixed if necessary. The lungs were instilled by the intratracheal route with 10 % neutral buffered formalin (20 cm water column) and then postfixed with the other organs in 10 % neutral buffered formalin. All slides were stained with haematoxylin and eosin (H&E) and with Prussian Blue.

Other examinations:

RECTAL TEMPERATURE:
Rectal measurements was made 5 rats/groups on days 0, 4, and 11. shortly after cessation of exposure using a digital thermometer equipped with a rectal probe for rats.

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variates three types of statistical test were used, the choice of the test being a function of prior knowledge obtained in former studies. The following statistical methods were used: Dunnet test, adjusted Welch test and Kruskal-Wallis test followed by adjusted U test.

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):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Iron oxide group:
- Main study: absolute and relative lung weight were statistically significant increased compared to the air control group (p < 0.01).
- Recovery study: relative lung weight was statistically significantly increased in the 1st postexposure period compared to the air control group (p < 0.01). At all time points of postexposure period, the organ weight of lung-associated lymph nodes (LALN) was statistically significant increased compared to air control group (p < 0.01).

Positive control (DQ12):
- absolute and relative organ weights of lung and LALN were statistically significantly increased in the main study and at all time points during postexposure period compared to the control group (p <0.01).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Exposed group: yellow discolouration of lungs and enlarged lung associated lymph nodes (LALN) were observed. Discolouration of the LALN was seen in all rats from this group at the end of exposure and during the whole recovery period (1 - 3 recovery period).
- Positive control (DQ12): grey discolouration of lungs was observed. All lungs from this group during all recovery necropsies were less collapsed upon opening of the thoracic cavity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Iron oxide group:
- Main study: at the end of the exposure period intraalveolar granular material was seen in all rats. These granules as well as the alveolar macrophages appeared to be yellow.
- Recovery study: at the end of the 13-week recovery period, incidence and intensity of focal inflammatory infiltrates and bronchiolar-alveolar hypercellularity decreased. Focal inflammatory infiltrates and focal septal thickening were detected in almost all animals. At all interim sacrifices iron oxide particles were detected in BALT.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID
Exposed group:
- At all time points the average recovery of the lavage fluid instilled into the lung was high, i.e., it exceeded 80 % of the instilled volume.

- Main study: statistically significant increases were observed in total cell count in BAL (TCC), mean cellular volume (MCV), lactate dehydrogenase (LDH), total protein (PROT) and β-N-Acetyl-glucosaminidase in BALF NAG compared to the air control group (p < 0.01 and p < 0.05).

- Recovery study: also, TCC were statistically significant increased in all recovery periods compared to air control group (p < 0.01).
The MCV level were also significantly increased in the first and second recovery period compared to air controls. There were statistically significant increases in the levels of LDH and PROT in the second and third recory period, and the mean cellular diameter (MCD) in the third recovery period, compared to air controls (p < 0.01).

- Positive control: DQ12 exposed animals were unequivocally positive at all time points with increasing intensity of changes during the course of the postexposure period. In the main study, statistically significant changes were observed in total cell count in BAL, LDH, AP, ACPH, total protein, and β-NAG compared to the control group (p < 0.01 and p < 0.05). These parameters, as well as the collagen and phospholipids in BALF were significantly increased at all time points during the postexposure period compared to the control group (p < 0.01 and p < 0.05).
For more information, please refer to table 1 in the field "attached background material" field below.

IRON DETERMINATION IN ORGAN TISSUES:
- Exposed group: a remarkable increase of iron was observed in lungs and in LALN with evidence of time related translocation from one compartment to another. The iron content in these organs were statistically significant increased at all time points in the postexposure period compared to the control group (p < 0.01 and p < 0.05).

IRON KINETICS IN LUNG AND LALN:
Assuming a single-compartment 1st order elimination kinetics from the lung, the elimination half-time for the test item was 75 days.

Details on results:

CLINICAL SIGNS:
- air control group: all rats tolerated the exposure without specific signs.
- Iron oxide group: Tachypnea (second exposure day only). As far as clinical signs were observed they were related to incidental and isolated findings without any time-related exacerbation.

MORTALITY:
No mortality occurred.

BODY WEIGHT AND BODY WEIGHT GAIN:
- there was no statistically significant difference in body weights amongst the groups.
- Iron oxide group: statistically significant changes in body weight gain were observed on days 0 – 4 and 63 – 70 in comparison to the air control group (p < 0.01).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS:
no significant changes were observed in the absolute and relative organ weights of brain, heart, thymus, liver, spleen, kidneys and testes in the main study and at all time points during postexposure period compared to the control group.

HISTOPATHOLOGICAL FINDINGS:
- At all interim sacrifices in the examined extrapulmonary organs (liver, kidneys, and testis) were no evidence of translocated iron particles existed. No iron oxide related findings could be detected in the liver, kidneys and testis at the end of the exposure period and after 2 weeks of recovery.

BALF MEASUREMENTS:
- Lipidperoxidation (TBARS) was measured in BALC and lung tissue and no distinct differences amongst the group was observed. Cytodifferentiation was planned however, omitted due to the extreme dust loading of BALC.

IRON DETERMINATION IN ORGAN TISSUES:
- Iron oxide group: there were no significant changes in the iron content of the liver and testes at all time points in the postexposure period compared to air control group. Splenic iron was examined on days 15 and 29. Due to a lack of any consistent effect and in the light of the spontaneously occurring variability in iron content, iron determinations in spleen were omitted on days 58 and 107.

RECTAL TEMPERATURE:
- Iron oxide group: in comparison to the concurrent air control group, there was no evidence of a conclusive, significant effect on body (rectal) temperature in the iron oxide group, whilst positive control (DQ12) elicited a hypothermic response on days 0 and 4.

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Conclusions:

In this RDT study, aerosolized geothite powder was administered by inhalation at a concentration of 195.7 mg/m3 to male Wistar rats. Exposure was through dynamic directed-flow noseonly for 6-hours/day on 5 days/week for 2 consecutive weeks. During a 3-month postexposure period, subgroups of rats (12 per sacrifice) were serially sacrificed 15, 29, 58, and 107 days and examined. DQ12 was served as reference dust (positive control for lung damage; MMAD (GSD): 2.27 µm (1.84)) in a concentration of 200.4 mg/m3. Body weights and clinical signs were recorded during the study and postexposure period. At each sacrifice inflammatory end points were determined in BAL, rats were examined for gross pathology, histopathology (lung, liver, spleen, kidneys, testes) and organ weights (lung, LALN, brain, heart, thymus, liver, spleen, kidneys, testes), and iron was determined in lungs, LALN, spleen, liver, and testes. aerosol was highly respirable to rats (MMAD: 1.31 μm; GSD: 2.16).
According to the author, 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 postexposure period. Conclusive evidence of bioavailable iron or iron particles that were translocated to extra- pulmonary organs was not observed. Extrapulmonary effects causally linked to the high-level exposure of iron oxide was not detected at any time point. At the end of the 3-month postexposure period the findings causally linked to the high-level exposure to iron oxides (e.g. focal inflammatory infiltrates, bronchiolo-alveolar hypercellularity) showed a decrease in incidence and/or severity.

This study was conducted as dose range finding study according to OECD 412 (1981) and under GLP.
However, only male rats were tested without justification, and only one dose was tested which does not allow a dose-response related analysis. A haematological examination and clinical biochemistry in blood were not conducted. Furthermore, the organ weight of the adrenals was missing, and a histopathological examination of adrenals and the heart were not performed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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. Three target substances covered by this read-across (Iron sinter; Iron ores, agglomerates; Mill scale) consist primarily of different iron oxides, as described in the technical dossier section 1.2 Composition. The predominant characteristic of the iron oxides 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 oral or inhalation exposure up to the limit dose of the maximum tolerated concentration of the respective test. Further information on the read-across approach is given in the report attached to IUCLID section 13.2. Iron is a transition-metal and is subject at its surface to passivation by the formation of a passive oxide (i. e. iron oxide) coating. In particular for iron metal and granules, the oxide layer will form a quantitatively continuous layer to envelop the entire particle irrespective of product form. In view of this, it may be assumed that human exposure is secondary to that of iron oxide and that the liberation of ionic iron shows a slower kinetics, compared with soluble iron salts.

 

Repeated dose toxicity - oral:

Human data – oral:

The human data of iron was obtained from publicly available peer-reviewed risk assessment documents, such as EFSA opinions, WHO recommendations for human nutrition. The references included in these risk assessment documents are not included in the dossier for the sake of brevity.

Considering the role of iron in human metabolic processes, it is highlighted that iron has several vital functions in the body. It serves as a carrier of oxygen to the tissues from the lungs by red blood cell haemoglobin, as a transport medium for electrons within cells, and as an integrated part of important enzyme systems in various tissues. It is an essential constituent of oxygen carriers, such as haemoglobin and myoglobin, and the iron contained within haem is essential for the redox reactions of numerous cytochromes. Insufficient intake results in the deficiency condition anaemia, adverse outcomes of pregnancy, impaired psychomotor development and cognitive performance and reduced immune function.

Any elemental iron in the diet is probably absorbed as non-haem iron following its dissolution in the acid stomach contents. The absorption of non-haem iron can be increased substantially by the presence of ligands, such as ascorbate, citrate and fumarate, as well as the presence of amino acids (e.g. cysteine) and oligopeptides resulting from meat digestion (Mulvihill et al., 1998). In contrast, very stable complexes, for example with phytates, phosphates and oxalates, impair non-haem iron absorption. Depending on the concentration of supportive or inhibitory ligands in the intestinal lumen the absorption of non-haem iron can vary by a factor of 10 in single-meal studies, but the effects are less pronounced in more long-term studies (Hallberg and Rossander, 1984; Rossander, 1987; Hunt and Roughead 2000). Iron is reversibly stored within the liver as ferritin and haemosiderin whereas it is transported between different compartments in the body by the protein transferrin. Iron excretion via the kidneys is very low, and body iron is highly conserved. Renal elimination is not controlled as part of iron homeostasis or the control of excess body stores. Normally, only about 0.1 mg is lost daily in urine. The sloughing of mucosal enterocytes results in elimination of absorbed iron before it reaches the systemic circulation and accounts for the loss of 0.6mg per day into the intestinal lumen. About 0.2-0.3 mg is lost daily from the skin. The total daily loss is equivalent to about 0.05 % of body iron content (Green et al., 1968).

The side effects of oral iron preparations increase with increase in dosage, but there are fewer side effects with slow delivery systems or if the iron is taken with food (Brock et al., 1985; Reddaiah et al., 1989). The adverse gastrointestinal effects are related to the concentration of iron in the intestinal lumen (Cook et al., 1990).

A daily dose of 50 mg of iron produced a higher incidence of gastrointestinal effects in subjects given conventional ferrous sulphate compared with subjects given the same amount in a wax-matrix (Brock et al., 1985), and also in subjects given ferrous sulphate compared with subjects given the same amount of iron as bis-glycino iron (Coplin et al., 1991). Neither of these studies included a placebo group, and therefore the association with iron is based on different responses to different preparations. A higher incidence of side effects was reported in subjects given 60 mg of iron as iron fumarate daily compared with placebo; daily doses of 120 mg of iron as fumarate for 8 weeks given to 19 young women in a double-blind cross-over study resulted in gastrointestinal effects in 5 subjects while receiving iron, compared with 2 while taking placebo (Frykman et al., 1994).

In a randomized double-blind, 3-wk trial by Gordeuk (1987) involving 36 female blood donors with mild Fe deficiency anaemia determined if high doses of oral iron could shorten the duration of therapy necessary to treat Fe deficiency anaemia, high-dose Fe 600 mg three times per day (1800 mg daily given as nontoxic carbonyl Fe) was compared with standard ferrous sulfate 60 mg Fe++ three times per day (180 mg Fe daily). a 10-fold increase in dose of Fe resulted in a 1.4- 1.6-fold increase in the positive Fe balance in the same period of time. High-dose carbonyl Fe was well tolerated with gastrointestinal side effects similar those observed with standard FeSO4 therapy. Both regimens corrected anaemia but neither replenished storage Fe. The author concluded that the principal advantage to the use of carbonyl Fe would derive from its safety rather than from the large doses that can be given.

 

References:

Brock C, Curry H, Hama C, Knipfer M, Taylor L (1985). Adverse effects of iron supplementation: A comparative trial of wax-matrix iron preparation and conventional ferrous sulfate tablets. Clin Ther 7: 568-573.

Cook JD, Carriaga M, Kahn SG, Schack W, Skikne BS (1990). Gastric delivery system for iron supplementation. Lancet 336: 1136-1139.

Coplin M, Schuette S, Leichtmann G, Lasher B (1991). Tolerability of iron: A comparison of bis-glycino iron(II) and ferrous sulfate. Clin Ther 13: 606-612.

Frykman E, Bystrom M, Jansson U, Edberg A, Hansen T (1994). Side effects of iron supplements in blood donors: Superior tolerance of heme iron. J Lab Clin Med 123: 561-564.

Reddaiah VP, Prasanna P, Ramachandran K, Nath LM, Sood SU, Madan N, Rusia U (1989). Supplementary iron dose in pregnant anemia prophylaxis. Ind J Pediatr 65: 109-114.

 

Animal data - oral

Iron oxide:

A sub-chronic 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 sub-chronic 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. The study is considered as reliable with restrictions (RL=2).

 

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. The study is considered as reliable without restrictions (RL=1).

 

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, 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 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. The study is considered as reliable without restrictions (RL=1).

 

Iron:

Whittaker, P. et al. (1997) describe the effects of micro sized carbonyl iron in rats and mice. The F344 rats and three strains of mice (B6C3F1, C5YSF1 yellow and C5YSF1 black) were investigated. Groups of 12 males per dose of the respective strain received the test substance at concentrations of 1500, 3500, 5000 and 10000 µg Fe/g in diet daily for 90 days. A control group receiving plain diet was also run concurrently.

In the RDT study with the male F344 rats, 9 of 12 rats died before the end of the 90-day treatment period at the 10000 µg Fe/g dose level. The body weights of rats in the 3500 - 10000 µg Fe/g group were reduced. There was a dose-related increase in liver non-haem Fe from the 3500 µg Fe/g group, and the Fe was stored in hepatocytes predominantly in the periportal region. Relative liver weights were dose-related and significantly increased in all dose groups. A significant hypertrophy of the hepatocytes was observed in rats fed 10000 µg Fe/g diet. There was also pancreatic atrophy with loss of both endocrine and exocrine tissue. In addition, rats in the 10000 µg Fe/g dose group had markedly exacerbated dose-dependent nephropathy and changes in glomerular and tubular epithelium associated with Fe accumulation. The rats also showed degeneration of the germinal epithelium of the testis, formation of multinucleated giant cells, and lack of mature sperm. Based on the effects observed in male F344 rats, the LOAEL was established at 1500 µg Fe/g diet (recalculation to the body weight: 68.07 mg Fe/kg bw/day).

In the RDT study by Whittaker et al. (1997) with the male B6C3F1 mice, no mortality and no effects on body weight gain were observed. There was a dose-related increase in liver non-haem Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. In addition, the relative liver weights were significantly increased in the 5000 and 10000 μg Fe/g dose groups. There was significant hypertrophy of the hepatocytes in mice fed the 10000 μg Fe/g diet. Morphometric evaluation of pancreas showed fewer β-cells. Based on the effects observed in male B6C3F1 mice, the NOAEL was established at 3500 µg Fe/g diet (recalculation to the body weight: 425.83 mg Fe/kg bw/day) and the LOAEL at 5000 µg Fe/g diet (recalculation to the body weight: 627.87 mg Fe/kg bw/day).

In the study by Whittaker et al. (1997) with the male C5YSF1 yellow mice, no mortality was observed. At all doses of Fe above the control, food intake, and body weight gain was reduced in mice. In all animals, there was a dose-related increase in liver non-haem Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. Morphometric evaluation of pancreas showed fewer β-cells in mice. There were fewer islets in the yellow C5YSF1 mice, and total and mean islet areas were smaller than in the control mice. Based on the effects observed in male C5YSF1 yellow mice, the LOAEL was established at 1500 µg Fe/g diet (recalculation to the body weight: 152.59 mg Fe/kg bw/day).

In the study by Whittaker et al. (1997) with the C5YSF1 black mice, also no mortality was observed. No effects on food intake or body weight gain were observed. The liver weights were increased in the 10000 µg Fe/g dose group. In all animals, there was a dose-related increase in liver non-haem Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. Based on the effects observed in male mice, the NOAEL was established at 5000 µg Fe/g diet (recalculation to the body weight: 604.4 mg Fe/kg bw/day) and the LOAEL at 10000 µg Fe/g diet (recalculation to the body weight: 1300.78 mg Fe/kg bw/day).

The four 90-day RDT studies by Whittaker et al. (1997) with the male F344 rats as well as male B6C3F1, C5YSF1 yellow or C5YSF1 black mice have the same several reporting and experimental deficiencies: The methods were not described in detail and not all parameters examined were presented in the results. Test material was insufficiently characterised (no purity, impurities). Only males were tested and the no. of animals per group (n=12) was too low for a statistical analysis. Animals were not observed for clinical signs, and cage-side observations were not performed. In addition, animals were weighed at end of study but not weekly during the study. Food consumption was only recorded at end of study but not weekly during the study. An ophthalmological examination, haematology or clinical biochemistry were not performed. The histopathological examination was incomplete since trachea, lungs, aorta, gonads, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, peripheral nerve, sternum with bone marrow were not examined. Information about test animals (source, acclimation period, initial body weight) and environmental conditions (photoperiod, temperature, humidity) were insufficiently. The results of the histopathological examination were not shown in tabular form, and the organ weights of heart, kidneys, brain, and thymus were not presented in the publication. Based on the above stated shortcomings, the study is considered as not reliable (RL=3) and used in a weight of evidence assessment for repeated dose toxicity via oral route.

 

Whittaker, P. et al. (2001) examined the effects of increasing levels of iron on serum fatty acids, cholesterol, triacylglycerol, liver and heart in groups of 8 - 10 male Sprague–Dawley rats. Rats were administered either with normal diet (contains 35 µg Fe/g (control group)), Fe-deficient or carbonyl iron supplemented diets at doses of 350, 3500 and 20000 µg Fe/g for 12 weeks. A control group receiving diet without supplementation of carbonyl iron and a plain diet were also run concurrently. Before termination of the study, 2 animals of the 3500 µg/g and 5 animals of the 20000 µg/g dose levels died. In both dose groups, the final body weights decreased compared to controls (35 µg Fe/g). The serum cholesterol levels and non-haem Fe levels in liver were increased in the 3500 and 20000 µg/g groups, as well as the non-haem Fe in heart of rats from the 20000 µg/g group. Furthermore, the relative heart and liver weights were increased in the 3500 and 20000 µg/g groups compared to controls. There were lesions in the heart of animals from these both dose groups. The incidence of severity of cardiomyopathy increased with higher dietary concentrations of Fe and was characterized by a spectrum of lesions. Five of the seven animals which died before study termination had heart damage, which included Fe in the cytoplasm of the myocardial fibres, haemorrhagic necrosis, epicardial damage and clot formation. In addition, in the liver of animals (6/10 rats) from the 20000 µg/g group hepatocellular hypertrophy was observed, manifested as enlargement of the individual hepatocytes. Based on the mortality, decreased final body weight, increased serum cholesterol, increased liver and heart weight, and the histopathological findings in heart (cardiomyopathy), the NOAEL was established at 350 µg Fe/g diet (recalculation to the body weight: 17.5 mg Fe/kg bw/day). This reference had several reporting and experimental deficiencies: The experimental set up and methods were not described in detail. Test material was insufficiently characterised (no purity, impurities). The actual concentration of iron in the diet was not analysed. A rationale for the selection of the dose levels was not given. The study duration (12 weeks) was too short for a sub-chronic RDT study. Only males were tested and the no. of animals per group (n=8-10) was too low for a statistical analysis. Based on the initial body weight of the males of approx. 42 g, an age of approx. 3 weeks is assumed before the start of the study (according to the growth chart of Sprague-Dawley rats). However, according to OECD 408 (1998), dosing should begin as soon as possible after weaning and the animals should be young healthy adults. If the animals were around 3 weeks old at the beginning of the study, that would be too young. At 3 weeks of age, they had not yet completed the weaning phase, which lasts 4 weeks for rats. Furthermore, animals were not observed for clinical signs, and cage-side observations were not performed. In addition, only the initial and final body weights were determined but the animals were not weighed weekly during the study. Food consumption was not recorded. An ophthalmological examination or haematology were not performed. During the clinical biochemistry, the levels of cholesterol and other lipids were analysed in serum, but the sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, creatinine, total protein and albumin, and more than two enzymes indicative of hepatocellular effects were not investigated. The histopathological examination was incomplete since only the liver and heart were examined. Information about test animals (no age or acclimation period) and environmental conditions (no photoperiod, temperature or humidity) were insufficient. A complete necropsy was performed but the results were not presented in the publication. The results of the histopathological examination were not shown in tabular form. Based on the above stated shortcomings, the study is considered as not reliable (RL=3) and used in a weight of evidence assessment for repeated dose toxicity via oral route.

 

No conclusion can be drawn from the below publications due to lack of quality, reliability and adequacy of the experimental data for the fulfilment of data requirements under REACH. The references represent in vivo experiments with investigations in repeated oral dose toxicity with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only:

Stal, P. et al. (1996): the publication shows significant methodological deficiencies in the experimental set up and documentation. Only male rats and only one dose group were tested which does not allow a dose-response related analysis. The number of animals per group (n=5) is not sufficient to perform an appropriate statistical analysis. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Only selected parameters (body weights at end of exposure, alcohol concentration in blood at study termination, histopathology of liver, and determination of iron content, protein, CYP2E1, MDA and NADPH dependent lipid peroxidation levels in liver) were investigated, and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Shelanski, H.A. (1950a): the publication shows significant methodological deficiencies in the experimental set up and documentation. Study duration was too short for a sub-chronic repeated dose toxicity study. The iron content of the diet for the control animals was not specified. Food consumption was not recorded, which makes it unclear how much iron was taken up with the diet. Not all animals in the group were examined for haematology, urinalysis and histopathology, and a statistical analysis were not performed. The number of animals per group (n=3-5) examined is not sufficient to perform an appropriate statistical analysis. Insufficient information about the test animals (strain and age were missing) and the environmental conditions (housing, temperature, humidity, lighting) are given.

Shelanski, H.A. (1950b): the publication shows significant methodological deficiencies in the experimental set up and documentation. Study duration was too short for a sub-chronic repeated dose toxicity study. Test material was insufficiently described (no purity). The iron content of the diet for the control animals was not specified. Food consumption was not recorded, which makes it unclear how much iron was taken up with the diet. Not all animals in the group were examined for haematology, urinalysis and histopathology, and a statistical analysis were not performed. The number of animals per group (n=3-5) examined is not sufficient to perform an appropriate statistical analysis. Furthermore, insufficient information about the test animals (strain and age were missing) and the environmental conditions (housing, temperature, humidity, lighting) are given.

Whittaker, P. et al. (1996): the publication shows significant methodological deficiencies in the experimental set up and documentation. The test substance is insufficiently characterised (no purity or impurities). Only male rats were tested. Study duration is too short for a sub-chronic repeated dose toxicity study. The number of animals per group (n=11-18) is not sufficient to perform an appropriate statistical analysis. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Food consumption was not recorded, which makes it unclear how much iron was taken up with the diet. Only selected parameters (histopathology, nonheme iron in liver and heart, iron content in different organs, lipid peroxidation) were investigated, and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

Whittaker, P. et al. (1992): the publication shows significant methodological deficiencies in the experimental set up and documentation. The test substance is insufficiently characterised (no purity or impurities). Only male rats were tested. Study duration is too short for a sub-chronic repeated dose toxicity study. The number of animals per group (n=10 or 11) for the investigations at the different time points is not sufficient to perform an appropriate statistical analysis. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Food consumption was not recorded, which makes it unclear how much iron was taken up with the diet. Only selected parameters (body weight gain, liver weight, nonheme iron in liver, weight of a piece from mucosa, lipid peroxides in liver and mucosa) were investigated, and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

 

Soluble iron salts:

Lynch et al. (2013) administered to groups of 10 male and 10 female Harlan Wistar rats an iron trichloride containing complexation/reaction product, termed FemTA by oral gavage. FemTA is a mixture of sodium tartrate [D(–)- and L(+)-tartaric acid and mesotartaric acid], sodium hydroxide, and iron trichloride. The composition of the product was approximately 4% sodium tartrate, 10% mesotartaric acid, 7% chloride, 4% iron, 7% sodium, 0.3% sodium oxalate, and 65% water. FemTA was administered to the groups at dose levels of 500, 1000, and 2000 mg/kg body weight/day (equivalent to 20, 40, or 80 mg of iron/kg body weight/day). Male rats were dosed prior to and during mating and up to the day prior to scheduled sacrifice during the post-mating period (total of 90/91 days). The females were treated with the substance prior to mating and during mating as well as during gestation and lactation (at least up to lactation day 4) (total of 104 to 109 d). During the treatment period the substance was administered once daily, 7 days per week. A control group was run concurrently. The study was conducted in accordance with OECD 422/408 and under GLP. During the observation of the parental (P) 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, behaviour (functional findings), and gross pathology. Compared to the vehicle control, treatment-related effects were observed in parental (P) rats receiving the substance at dose levels of 1000 and 2000 mg/kg body weight/d. During the haematological examination, an increase in white blood cell count (p < 0.01) and relative neutrophil counts (p < 0.05) as well as a decrease in relative lymphocyte count (p < 0.05) were noted for male rats of the 2000 mg/kg bw/day dose level. Furthermore, an increase in relative eosinophil counts (p < 0.05) were observed in females at the 2000 mg/kg bw/day dose level. Also, treatment-related effects were observed for clinical biochemical findings. At the 1000 and 2000 mg/kg bw/day dose levels, increased blood urea nitrogen (p < 0.01) and bile acid levels (p < 0.01) were noted for male rats. Furthermore, an increase in alanine aminotransferase (p < 0.01) as well as a decrease in sodium and chloride concentrations were observed in male rats at the 2000 mg/kg bw/day dose level. Increased blood urea nitrogen (p < 0.05) and decreased chloride concentrations (p< 0.05) were recorded for female rats at the 2000 mg/kg bw/day dose level. At the 500, 1000 and 2000 mg/kg bw/day dose levels, an increased absolute (p < 0.05) and relative kidney weight (p< 0.01) was observed in the male rats of the parental generation. In addition, an increased absolute (p < 0.05) and relative liver weight (p < 0.01) was recorded for male rats of the 2000 mg/kg bw/day dose level. At the 1000 and 2000 mg/kg bw/day dose levels, elevated organ weights were observed for absolute (2000 mg/kg bw/day dose level only; p < 0.01) and relative kidney weights (p < 0.01) for female rats of the parental generation. Alterations in kidney weight were clearly treatment-related, and, given the magnitude, were considered adverse at the 2000 mg/kg body weight/d dose level. Treatment-related effects were observed during microscopical examination of the parental generation. Inflammation of the gastro-intestinal tract (increase in both incidence and severity of neutrophilic infiltrates, acute inflammation, goblet / epithelial cell hyperplasia, foci of brown pigment and/or oedema of the rectum, colon and cecum) was observed at the 1000 and 2000 mg/kg bw/day for both sexes and this finding was considered to be treatment-related.

Based on the histopathological findings noted in the gastro-intestinal tract of male and female rats of the parental generation at the 1000 and 2000 mg/kg bw/day dose levels, the no observed adverse effect level (NOAEL) for general toxicity is 500 mg/kg bw/day for males and females. The study is considered as reliable with restriction (RL2). The following experimental and recording deficiencies can be reported for the publication: test substance is a mixture, therefore, it is unclear, if the observed effects were due to the iron content of the mixture alone; mixture has a low pH (3.7), which might cause the observed effects in the intestine; exposure duration was not clearly described; exposure duration to long; clinical signs were not fully described; detailed clinical examinations were not fully described; historical control data and individual data missing.

 

In a combined repeated dose toxicity study with the reproduction developmental toxicity screening test (published study report by the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Japan, 2002), groups of 12 male and 12 female Sprague-Dawley rats were administered iron sulphate heptahydrate via gavage at dose levels of 30, 100, 300 and 1000 mg/kg bw/day (equivalent to 6, 20, 60 and 201 mg Fe/kg bw/day). Males and females were treated with the substance for a duration of 49 days (14 days before mating and 35 days after mating) and 42 - 47 days (14 days before mating, throughout the mating period and the gestation period until lactation day 5), respectively. A vehicle control group was run concurrently. The study was conducted in accordance with OECD 422 and under GLP. After oral administration of 30, 100, 300 and 1000 mg/kg/day of the test item no effects were observed on food consumption and gross pathology. General observation revealed salivation in males and females in the ≥ 300 mg/kg groups. This was transient and only observed immediately after administration, and there were no neurological symptoms such as convulsion or morphological changes to the salivary glands, and so the salivation was attributed to irritation by the test substance and was not deemed to be a symptom of toxicity. After the administration 1000 mg/kg/day of the test item, one male and one female died. These animals had exhibited salivation on observation of general condition. Necropsy of the dead animals revealed adrenal hypertrophy in the male and pituitary tumour, atrophy of the thymus, dark red discolouration of the lungs and adrenal hypertrophy in the female. Histological examination revealed mineral deposition in the heart, congestion of the lungs and yellow-brown pigment deposition in the periportal hepatocytes in the male and congestion and oedema in the lungs and mineral deposition in the liver of the females. In addition to the findings described above for the 1000 mg/kg/day dose group, body weights in the 1000 mg/kg group were somewhat low throughout the administration period in the males and tended to be low in the late gestation period in the females. Furthermore, temporarily low food consumption was observed in males and females in this group. Urine tests revealed high urine volume and low specific gravity in males of the 1000 mg/kg/day group, but no changes attributable to test item administration were observed in the females. Haematology tests revealed low red blood cells (RBC) and activated partial thromboplastin time (APTT) values, and high mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and reticulocyte levels in males, but no changes attributable to administration were observed in the females. Blood biochemistry test revealed low total protein, albumin and Ca levels, and high Alanine transaminase (ALT), γ-Glutamyl transferase (γ-GTP) and albumin/globulin (A/G) levels in males and high γ-GTP and organic phosphorus levels in females. The necropsies revealed dark red spots and ulceration of the glandular stomach mucosa in males in the 1000 mg/kg group, but no changes caused by administration were observed in the females. Further, organ weight measurements revealed high absolute and relative adrenal weights and high relative liver weights in males in the 1000 mg/kg group, and high absolute and relative liver weights in females in the 1000 mg/kg group. Lastly, the histological investigation of the 1000 mg/kg/day group revealed that the thymus findings were atrophy of the thymus in two males. The stomach findings were ulceration of the glandular stomach in one male, erosion of the glandular stomach in one male, inflammatory cell infiltration of the glandular stomach submucosa in two males, haemorrhage of the glandular stomach submucosa in one male, and vacuolisation of the forestomach epithelium in one male. The liver findings were yellow-brown pigment deposition in periportal hepatocytes in all six males, and yellow-brown pigment deposition in periportal Kupffer cells in three males and yellow-brown pigment deposition in periportal hepatocytes in all six females. The spleen findings were extramedullary haematopoiesis in four males, and yellow-brown pigment deposition in the red pulp in all six males and yellow-brown pigment deposition in the red pulp in all six females. These findings were observed at greater severity in the high dose group than in the control group. The kidney findings were basophilic changes in the tubular epithelium in four males. The bone marrow findings were increased haematopoiesis in the femur in one male. High organic phosphorus levels were seen in females of the 300 mg/kg group. Furthermore, the histopathological investigation revealed increased extramedullary haematopoiesis in the spleen in five males. Furthermore, no histopathological changes were observed in the testes, epididymis, seminal vesicles, prostate, ovaries, uterus, vagina or mammary glands in parental animals at any dose level. In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of 300 mg/kg/day (equivalent to 60 mg Fe/kg bw/day) is derived for both sexes of the parental generation based on the increased relative liver weight and increased gamma glutamylpeptidase in males and females at the 1000 mg/kg/day dose level. The study is considered as reliable with restriction (RL2), details on the shortcomings are reported in the IUCLID study record. The following deficiencies can be reported for the publication: the study Males were not dosed during mating. The length of mating period was not clearly stated. Detailed clinical observations & investigations of neuro-behaviour were missing. Potassium in blood serum was not measured. Historical control data are missing. The study is considered as reliable with restrictions (RL=2).

 

Rao and Jagadeesan (1995) describe the developmental of a rat model for iron deficiency and toxicological studies. Three strains of rats (Fischer-F 344-N, Sprague Dawley, and Wistar/nin) were investigated. Groups of 10 animals of each strain received an iron-deficient diet (< 10 ppm; equivalent to < 1.2 mg/kg bw/day) ad libitum for a duration of six weeks. Furthermore, a control group of ten animals of each strain received a diet supplemented with ferrous sulphate which contained an iron content of 220 ppm (equivalent to 26.4 mg/kg bw/day). Clinical signs of iron deficiency were evident in the Fischer and Wistar experimental rats. Fur growth was not normal, and their eyes were pale. Furthermore, body weight of iron-deficient Fischer 344 and Wistar strains (experimental groups) was reduced 20 - 30 % compared with control rats. The Sprague Dawley iron-deficient rats did not have any difference in body weight compared with their controls. In addition, food consumption of Fischer 344 and Wistar rats fed the iron-deficient diet (experiment groups) was decreased 25 - 30 % compared with those fed the control diet during the experimental period. This decrease was not evident in the Sprague Dawley iron-deficient rats. These differences in food consumption were reflected in body weight of the three rat strains. Feed efficiency was reduced to 80 % in the Fischer 344 iron-deficient rats and to 65 % in the Wistar iron-deficient rats (experiment groups). Feed efficiency of Sprague Dawley iron-deficient (experimental group) and control rats was similar. Comparison of the mean values for control and iron-deficient groups within each strain indicated that all haematologic variables such as haemoglobin concentration, protoporphyrin-to-heme (P/H), and liver and serum iron concentrations were significantly affected by iron deficiency (experimental groups). However, the magnitude of changes was not the same among various strains. The Fischer strain was the most susceptible to iron deficiency. It had the lowest haemoglobin concentration compared with the other two strains (5.37 versus 6.12 versus 7.42 g/dl, Fischer versus Wistar versus Sprague Dawley respectively; all values were significantly different from each other). Further, serum and liver iron values were lowest, and the P/H was highest in Fischer rats compared with the other two strains. The increase in the P/H value confirms development of iron deficiency which, however, was not significant among groups because of small sample size and large variations. Reduction in serum but not liver iron concentration was significantly different among the strains. Lastly, gross pathological findings were evident in Fischer and Wistar rats receiving the iron-deficient diet (experimental groups). Internally, the liver was pale, the colon and stomach were distended, and the heart and spleen were enlarged. This reference shows several reporting and experimental deficiencies: Firstly, the test item was insufficiently characterised, which makes it impossible to verify the identity of the test item. Furthermore, male rats were only investigated, which makes it impossible to draw any conclusion on the effect iron-deficient diet has on females. In addition, three dose levels were not investigated as foreseen by the OECD guideline. Therefore, a dose-response related analysis cannot be conducted, and a No-Observed-Adverse Effect level (NOAEL) cannot be determined. Clinical signs were recorded, but no indication was made on the time schedule used for recording. Furthermore, the authors did not mention, if they recorded/observed any mortality. The table presenting the haematologic parameters does not included all animals (n = 10/group), but there is no explanation why they were excluded. This raises the question if these animals did not survive until the end of the study. Also, the haematological examination (haemoglobin concentration, protoporphyrin-to-haem ratio and serum iron only) as well as the clinical chemistry examination (liver iron concentration only) were incomplete. In addition, organ weights were not determined, and histopathology of the animals was not conducted. Lastly, individual data and historical control data were missing. Since individual data is missing, it makes it impossible to observe any outliners that might influence the outcome of the study. Since the historical control data is missing, it cannot be determined, if the results observed were not within the normal biological variation of the strains investigated and, therefore, a treatment-related effect is observed. Based on the above stated shortcomings, the study is considered as not reliable (RL=3) and used as supporting information only.

 

Kim et al 2004 and Bae et al. 2005 report a combined repeated dose toxicity study with the reproduction/developmental toxicity screening, which was conducted in accordance with OECD 422 and under GLP. Groups of 15 male and 15 female Sprague Dawley rats were administered via gavage iron dichloride at dose levels of 125, 250 and 500 mg/kg bw/day (equivalent to 55, 110 and 220 mg Fe/kg bw/day). The male and female rats were treated daily for a duration of 42 days and 42 to 54 days, respectively. The treatment period included a two-week pre-mating period as well as mating period, gestation period and lasted up to lactation day 4. A control group without treatment was run concurrently. In addition, recovery groups of five male and five female rats were employed for the control group and the high dose group. During the observations of the parental male and female rats, no test item-related effects were observed for food consumption, haematology, clinical biochemistry, urinalysis, neuro-behaviour). However, clinicals signs such as blackish stool and salivation were observed for both sexes at the 125, 250 and 500 mg/kg bw/day dose levels. Furthermore, in the early stages of administration of 500 mg/kg bw/day, cases of decrease in locomotion activity were found in both sexes, but these were recovered to normal states. The female rats were more sensitively affected than the male rats in locomotion activity decrease, paleness, emaciation and soiled perineal region. However, these symptoms were reversible within the test period. In addition, three female rats of the 500 mg/kg bw/day dose group were found dead on the days 38, 46 and 51. The cause of death was gastrointestinal damages by the test substance. At the 250 and 500 mg/kg bw/day dose levels, the rate of body weight gain of the parental male rats was significantly decreased (- 4.2 % to - 8.2 % and - 7.5 % to -14 %, respectively) compared to the control group and water consumption was increased for male and female parental animals at the 500 mg/kg bw/day dose level compared to the control group. The investigation of the organ weights of the parental generation revealed that both absolute and relative weights of liver were statistically significantly increased in 250 and 500 mg/kg bw/day male groups and in 500 mg/kg bw/day female group. Also, for male rats, absolute adrenal glands weights were statistically significantly increased in 500 mg/kg bw/day group, and relative adrenal glands weights were increased in 250 and in 500 mg/kg bw/day group. Because of hemosiderin deposit in hepatocyte and hyperplasia of zona fasciculate in adrenal cortex, the increased weights of liver and adrenal glands were influenced by the test substance. In 125 mg/kg bw/day male group, liver weight did not differ from the control group, but adrenal glands weights (left) were decreased as compared to the control group. Furthermore, the following necropsy findings were caused by the test substance: severe diffuse haemorrhagic glandular stomach and severe distension of stomach in dead animals, and diffuse black coloured liver and haemorrhage with diffuse black pigmentation in scheduled necropsy of 500 mg/kg bw/day male group. For females, a case of mass of mesenteric lymph node was observed in 500 mg/kg bw/day group. Lastly, the histopathological examination showed that for groups of both sexes, hemosiderin deposit of hepatocyte and glandular, hyperplasia of zona fasciculate in adrenal cortex, hyperkeratosis of forestomach, hemosiderin deposit of glandular stomach, neutrophil infiltration of submucosa were observed at the 500 mg/kg bw/day dose level. These conditions were induced by the test substance and were weaker in females. There were no specific findings in the recovery groups. Based on the test item-related effects on body weight gain, water consumption, organ weights (liver and adrenal gland), macroscopical findings and microscopical findings, the no observed adverse effect level (NOAEL) for general toxicity is 125 mg/kg bw/day for males. The NOAEL for general toxicity is 250 mg/kg bw/day for females based on mortality, water consumption, organ weight (liver) and microscopical findings. The study results were only available in a brief publication by Bae et al and as extended study summary (including raw data) in the OECD SIAR for Iron. The original reference was not obtainable, but the study was approved by the OECD procedure on Mutual Acceptance of Data (MAD). However, the reporting detail is insufficient so that the study was rated with reliability 4. Further details are reported in the IUCLID study record.

 

Triiron phosphide:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (unpublished report by O’Halloran (2012)), iron phosphorous (Fe3P) in 1 % (w/v) aqueous methylcellulose was administered via gavage 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 five consecutive weeks or daily for two weeks before pairing throughout mating, gestation and until lactation day 6 for males and females, respectively (main phase groups). A vehicle control group was run concurrently. In addition, groups of 5 females were administered the substance at dose levels of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day via gavage for five consecutive days and these females were not mated (toxicity phase groups). During the observations of the parental animals, no test item-related effects were observed for clinical signs, mortality, body weight gain, food consumption, haematology, clinical biochemistry, neuro-behavioural examinations, organ weights, gross pathology and histopathology. Based on the results from this study, for general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be greater than 1000 mg/kg/day. The study was performed according to OECD test guidelines, and in compliance with GLP, so the data is considered reliable without restriction (RL=1).

 

Summary entry – Repeated dose toxicity: oral

Another eight references were also identified, representing in vivo experiments with investigations on repeated dose oral toxicity. These experiments were conducted with mice or rats receiving carbonyl iron, electrolytic or reduced iron and diiron trioxide via oral (gavage or unspecified) administration. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. The references usually lack significance due to, e.g., poor test item characterisation, low number of animals used, missing dose response relationship, or unjustified dosing regime. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given below were included in the IUCLID for information purposes only:

Van Wyk, C.P. et al. (1974): only male rats were tested. The number of animals per group (n=8) is not sufficient to perform an appropriate statistical analysis. The experimental design is not in accordance with any relevant guideline. Only selected parameters (iron intake and non-haemoglobin iron in rat liver) were investigated, and the investigated endpoints are not required for building an expert judgement and further assessment of the test substance under REACH (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X).

 

Conclusion - oral

The iron oxides, show a complete absence of adverse effects in guideline compliant repeated dose toxicity studies via the oral route. Neither macroscopic, nor microscopic findings were noted. These findings were seen in a sub-chronic oral study in rats with three different iron oxides. There was also an absence of adverse effects in a sub-acute oral study in rats with triiron phosphide. A NOAEL was not derived based on the absence of adverse effects up to the limit dose of 1000 mg/kg bw/day.

A number of sub-chronic oral repeated dose toxicity studies with iron in different species were conducted by the same working group. The studies show a substantial variability in the effect doses (NOAEL range from 68 to 1300 mg Fe/kg bw/day, with a mean of 390 mg Fe/kg bw/day), which may be caused by the poor reporting quality and deficiencies in the study design. The authors describe the effects as mild increase in relative liver weight and increase in liver non-haeme Fe. In a double-blind clinical trial some very mild effects are described after treatment of anaemic patients with iron (powder) as food supplement at 1800 mg Fe/day for three weeks. The treatment was generally well-tolerated with transient gastrointestinal side effects comparable to the effects seen with soluble iron salts. A comparative group of patients was treated with a soluble iron salt at 180 mg Fe/day, very similar effects were observed compared with the iron-treated group. This data shows an approx. 10-fold lower toxicity for iron compared with soluble iron salts.

For information purposes and for comparative purposes, toxicological data on soluble iron salts were included in this dossier as follows: A substantial data base of short-term animal studies as well as human data on the treatment of anaemic patients with soluble iron salts (e.g. iron sulphate, iron chloride and iron tartrate) is available. The sub-acute animal studies indicate an effect dose of 60-180 mg Fe/kg bw/day based on increased liver weights and inflammatory parameters in the gastro-intestinal tract. The effects in the gastro-intestinal tract are also identified as lead-effect in the human studies, where effect levels of 60-200 mg Fe are described (see discussion above).

The lower effects levels for highly soluble and highly bioavailable iron substances compared to the higher effect level for iron for both in animals and humans, corroborates the assumption that solubility in water or artificial body fluids correlates with the systemic toxicity of the iron category substances. This is further confirmed by the complete absence of adverse effects in three sub-chronic repeated dose toxicity studies in rats with iron oxides (which represent the least soluble and bioavailable iron category substances). Further details on the read-across is provided in the report attached to IUCLID section 13.2.

 

Repeated dose toxicity – inhalation:

Human data - Cohort studies

Axelson, O. and Södberg, A. (1979) studied in a case-control study the cancer incidence in a worker population of 80000 (≥ 20 – 75 years old) who were highly exposed to dust of iron oxides, particularly hematite, and with some impurities of pentavalent arsenic and other metals during a period of 1959 – 1971. Despite the high exposure levels, no excess of cancer had been observed, either in the respiratory system or at other sides. The rate ratio (RR) for lung cancer was 0.63 (CI 0.29-1.34) for exposed compared to unexposed. The expected lung cancer incidence was about that expected for an urban population in comparison to the national average. The results indicate no association between iron oxide exposure and any of the various types of cancer. Based on the weaknesses of this study (small number of exposed workers, 10 years latency period without knowledge of smoking habits, assumed workroom air level of iron oxide - no measurement, impurities of iron oxide (1-2 % of copper, small amounts of arsenic, nickel, and cobalt)) this reference had reporting deficiencies which renders it of very limited value for human health risk assessment of iron oxide; merely of supportive information use. No conclusion can be drawn from the publication due to a lack of quality, reliability, and adequacy of the data for the fulfilment of data requirements under REACH.

 

Bourgkard, E. et al. (2009) assessed in a case-control study the possible association between iron oxide exposures and lung cancer mortality among 17701 male and female workers in a French carbon steel-producing factory, taking into account the main possible occupational (PAHs, silica, asbestos) and non-occupational (smoking) confounders. The occupational exposures were assessed by a specific job-exposure matrix (JEM) through the subjects’ job histories, developed by a group of eight experts (two epidemiologists, two hygienists and four physicians). The mortality rate of the cohort was compared with local (département du Nord) and French death rates as external reference using standard life tables, furthermore internal comparisons were conducted to base on qualitative and quantitative exposure parameters as assessed by the JEM. The present study did not detect any relationship between exposure to iron oxides and lung cancer mortality. An excess of mortality from bladder cancer was found among workers exposed to oil mist. Besides the weaknesses of this study (mixed exposure and missing smoking habits of the workers during the 10-years lag period, missing informed consent) this reference can be used for human health risk assessment but merely as supportive information.

 

Moulin, J. et al. (2000) assessed in a cohort study the risk of lung cancer due to exposure to iron, chromium, nickel and/or their compounds, and took into account possible confounding by smoking and exposure to other known occupational carcinogens. It consisted of a cohort study with 4288 male and 609 female subjects followed by a nested case-control study (instead of a full cohort analysis for reasons of efficiency), and occupational exposure was assessed using a specific job-exposure matrix (JEM) developed by a panel of experts. This study failed to detect any relationship between lung cancer and exposure to iron. High and statistically significant relative risks, along with increasing trends, were observed for simultaneous exposure to PAHs and silica. These relationships were not confounded by smoking. Based on the weaknesses of this study (mixed exposure and missing smoking habits of the workers during the 10-years lag period, missing informed consent) this reference cannot be used for human health risk assessment but merely as supportive information.

 

Summary entry – repeated dose inhalation toxicity

Another forty-three references were identified during a literature search, representing investigations on repeated dose inhalation toxicity. These investigations were conducted with occupationally exposed workers to iron oxides. The references usually lack significance due to, e.g., insufficient description of method, uncertainty in number workers analysed and/or confounding factors cannot be excluded. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given below were included in the IUCLID for information purposes only:

Buerke, U. et al. (2002): The co-exposure to other elements (Cr, Ni, Mn, Al, Cu, Ti, SS) besides iron oxide in the welding fume and the different smoking habits of the welders are unknown.

Charr, R. (1955): Exposure parameters (composition and amount of welding fume) and the smoking habits of the most welders are unknown.

Charr, R. (1956): Exposure parameters (composition and amount of welding fume) and the smoking habits of the welders are unknown.

Funahashi, A. et al. (1988): Exposure parameters (composition and amount of welding fume) and the smoking habits of the welders are unknown.

Groh, J.A. (1944): Exposure parameters (composition and amount of welding fume) and the smoking habits of the welders are unknown.

Guidotti, T.L. et al. (1978): Exposure parameters (composition and amount of welding fume) and the smoking habits of the welder are unknown.

Kleinfeld, M. et al. (1969): The composition of the welding was not specified, thus relevance cannot be judged.

McCormick, L.M. et al. (2008): The amount and composition of the welding fume is unknown.

Näslund, P.E.; Högstedt, P. (1982): the composition and duration of exposure of the welding fume were not specified.

Offermann, P.V.; Finley, C.J. (1992): The amount and composition of the welding fume (welding with galvanised steel and inhalation of carbonyl fumes) are unknown.

Patel, K.C. et al. (1977): The amount and composition of the welding fume, the simultaneously exposition to asbestos and the unknown smoking habits were not specified.

Stern, R.M. et al. (1988): The composition of the welding fume was not described, thus relevance cannot be judged.

Sander, O.A. (1947): exposure parameters (composition and amount of welding fume), the smoking habits and sex of the oxyacetylene cutters were not specified.

Mann, B.T.; Lecutier, E.R. (1957): The subjects were exposed to different mixtures of several compounds during the welding process. The smoking habits of the arc welder were not specified.

Jones, J.G.; Warner, C.G. (1972): The subjects were exposed to different mixtures of several compounds during the welding process.

Harding, H.E. et al. (1958): The subjects were exposed to different mixtures of several compounds during the welding process. The smoking habits of the cases are missing.

Hamlin, L.E. et al. (1950): The subjects were exposed to different mixtures of several compounds in the fumes and dusts.

Doig, A.T.; McLaughlin, A.I.G. (1936): The subjects were exposed to different mixtures of several toxic compounds of arc welding fumes. The smoking habits of the welders are missing.

Doig, A.T.; McLaughlin, A.I.G. (1948): the subjects were exposed to different mixtures of several toxic compounds of arc welding fumes. The smoking habits of the welders were not specified.

Attfield, M.D.; Ross, D.S. (1978): the subjects were exposed to a mixture of several toxic compounds (Mn, Ni, Cr, Zn, Pb, nitrogen oxide and asbestos). The fume level concentration and the smoking habits of the welders were not specified.

Enzer, N.; Sander, O.A. (1938): Individuals were exposed to different mixtures without amount and concentration measurement. The smoking habits of the welders were not specified.

NIOSH (1976): The welders were subjected to mixed exposure and information on confounding factors such as smoking habits are missing.

NIOSH (1983): This study checked if the dust concentration of iron oxide in a plant were below or above (ACGIH TLV's 1981). No direct results (e.g., clinical signs, histology) for human health were stated. The welders were subjected to mixed exposure, and the smoking habits are not described.

NIOSH (1977): The welders were subjected to mixed exposure, and the smoking habits were missing.

Fawcitt, R. (1943): The exposure to radon and diesel exhaust gas is unknown. Amount and composition of mixed dust exposure and smoking habits are not described. A counting and statistical analysis of cases and diagnoses were not performed.

Morgan, W.K.C. (1978): Confounding exposure to cigarette consumption (smoking of 1/2 a pack cigarettes per day), unclear aetiology of the health effects.

Moore, E. et al. (1987): Individuals were co-exposure with 13 % quartz and 40 % iron.

Kleinfeld, M. et al. (1968): miners and sinters were subjected to mixed dust exposure (silicates, and free silica), and there was a possible exposure to other agents (radon, ozone, nitrous oxides, or substances in the welding fume).

Stewart, M.J.; Faulds, J.S. (1934): the exposure was a mixture of iron, silica and other components. The exposure parameters, and smoking habits are not described.

Boyd, J.T. et al. (1970): The iron-ore miners were exposed to radon, radon daughters, diesel exhaust gas, and were thus exposed to a mixed dust. The smoking habits ware unknown.

Chen, S. et al. (1990): The iron-ore miners were also exposed to a mixed dust of radon, radon daughters, diesel exhaust gas.

Edling, C. (1982): the iron-ore miners were exposed to radon, radon daughters and diesel exhaust gas. The amount and composition of mixed dust exposure were not known.

Jorgensen, H.S. (1973): the subjects were exposed to dust mixtures of hematite, radon and radon daughters. The smoking habits were known but not taken into account for the calculation.

Mur, J.M. et al. (1987): the smoking habits for 70 % of miners is not specified. The iron-ore miners were subjected to a co-exposure, and the amount of exposure was not specified.

Lawler, A.B. et al. (1985): Smoking habits of miners were not specified. The amount, and composition of mixed dust exposure are not described, thus relevance cannot be judged.

Harding, H.E. (1948): The subjects were exposed to a mixture of iron oxide and silver dust (silica). The smoking habits of the silver finisher are missing.

Barrie, H.J.& Harding, H.E. (1947): The silver finishers were subjected to mixed exposure to iron oxide and silver dust. The smoking habits of the silver finisher are not described.

McLaughlin, A.I.G. et al (1945): The silver finishers were subjected to mixed exposure of iron oxide, silver and cotton dust. The smoking habits of the silver finishers are not described.

Buckell, M. et al. (1946): the iron tuner and grinders subjected to mixed exposure of different compounds. The amount of exposure and the missing smoking habits of the iron tuner and grinders are not described.

Pendergrass, E.P.; Leopold, S.S. (1945): Grinders were subjected to mixed exposure of the grinders dust. The smoking habits of the grinders were not specified.

Teculescu, D.; Albu, A. (1973): The authors stated that the dust contained only pure iron oxide, although mixed exposure cannot be excluded due to the manufacturing process of red iron oxide, which used iron carbonate, natrium carbonate and iron sulphate.

Turner, H.M.; Grace, H.G. (1938): The components and amounts of mixed exposure for each occupation are missing. The smoking habits of the subjects are missing.

Goldberg, M.S. et al. (2001): There are no data on co-exposure to other agents, and on exposure parameters. The exposure to iron oxide was estimated from job description.

 

 

Animal data

In a subacute inhalation toxicity study, 48 male Wistar 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). 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. This supports the conclusion, that Fe3O4 can serve as a surrogate for FeOOH and Fe2O3. The study is considered as not reliable (RL=3) and used in a weight of evidence assessment for repeated dose toxicity via inhalation route.

 

For Fe3O4 valid subacute and sub-chronic inhalation studies are available (Pauluhn, 2006a; Pauluhn, 2006b; Pauluhn, 2011). In the subacute inhalation toxicity study 30 male Wistar 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 sub-chronic 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 haematology, 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. Haematology, 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 sub-chronic exposure in rats (Pauluhn, 2006a; Pauluhn, 2006b). The study by Pauluhn (2006a) was performed according to OECD test guidelines, and in compliance with GLP, so the data is considered reliable without restriction (RL=1). The study by Pauluhn (2006b) is considered as reliable with restrictions (RL=2) based on missing haematological or clinical biochemistry analyses.

 

In a short-term repeated dose toxicity study, Warheit, D.B. et al. (1997) investigated the pulmonary effects of aerosolized carbonyl iron in male Crl:CDBR rats after 4 weeks of exposure and 6 months of postexposure. Groups of 10 rats were exposed to micro-sized carbonyl iron particles 6 hours/day, 5 days/week via nose-only inhalation at mean actual concentrations of 5, 50, and 250 mg/m³.

Exposure to high dust concentrations of carbonyl iron particles produced sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, deficits in macrophage function, and the appearance of macrophage aggregates at sites of particle deposition. Based on the lesions in the respiratory tract observed at exposure concentration of 50 and 250 mg/m3, and the increased LDH and protein values at 250 mg/m3, the NOAEC was established at 4.8 mg/m3 (actual concentration). This reference had several reporting and experimental deficiencies: The publication shows significant methodological deficiencies in the experimental set up and documentation. Test material was insufficiently characterised (no purity or impurities). Only male rats were tested. The type of inhalation exposure was not specified. The method for verification of carbonyl iron concentrations in the exposure air was not described. Based on the above stated shortcomings, the study is considered as not reliable (RL=3) and used in a weight of evidence assessment for repeated dose toxicity via inhalation route.

 

In another short-term repeated dose inhalation study, Warheit, D.B. et al. (1991) investigated the pulmonary response of micro-sized carbonyl iron particles in male rats (Crl:CD BR) after short-term nose-only inhalation exposure. Groups of 30 rats (6 per time point for analyses) were exposed to the carbonyl iron aerosol (MMAD 3.6 µm) at a mean actual concentration of 110 mg/m³ for 6 h/day over a total period of 3 days via nose-only inhalation. A control group of 98 rats (14 per time point for analyses) inhaling air was also run concurrently. The lung burden in carbonyl iron-exposed rats was estimated to be 620 µg iron/lung. Short-term inhalation exposure did not induce significantly altered BALF parameters at any time point, when compared to the sham control group. The airway and parenchymal cell turnover rates were not significantly different from controls. Histopathological examination did not reveal any pulmonary lesions at any time post exposure. Due to the unsuitable study design with the following major restrictions this study will not be used for hazard and risk assessment purposes but as supplementary mechanistic information: This study was not in accordance with any subacute inhalation toxicity guideline. The purity of the test material was not specified. Male rats were used for the toxicity evaluation, only. The number of animals per group and time point (n = 6) was low, and thus, robustness is only limited. A justification for the concentration tested is not provided. The use of only one dose group precludes dose-response relationship evaluations. Details on the test animals (e.g. weight at study initiation and test group randomisation) and housing conditions (e.g. environmental conditions and group sizes) are missing. The investigations are restricted to pulmonary effects. Information on food consumption, body weight development, clinical chemistry, haematology, and histopathology results of other organs than lung is missing. Methodology on lung burden analysis and aerosol generation is insufficiently described. Based on the above stated shortcomings, the study is considered as not reliable (RL=3) and used in a weight of evidence assessment for repeated dose toxicity via inhalation route.

 

Summary entry – Repeated dose toxicity: inhalation

Another three references were also identified, representing in vivo experiments with investigations on repeated dose inhalation toxicity. These experiments were conducted with rats or guinea pigs receiving hematite, diiron trioxide or carbonyl iron via inhalation (whole body, head-nose or nose-only). The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. The references usually lack significance due to, e.g., poor test item characterisation, low number of animals used, missing dose response relationship, or unjustified dosing regime. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given below were included in the IUCLID for information purposes only:

Haynes, F. (1931): the type and composition of haematite dust was not reported, exposure conditions were poorly defined. The publication shows significant methodological deficiencies in the experimental setup and documentation (test material was not characterised, exposure concentration and method of aerosol generation were not specified). Only selected endpoints (histopathology of lungs and trachea) were investigated.

Ma-Hock, L. (2015 (A)): the study duration of 5 days is too short for a short-term repeated dose toxicity study. Only male rats were tested. Only two dose groups were examined which does not allow a dose-response related analysis. The number of animals per group (n= 3-5) is not sufficient to perform an appropriate statistical analysis.

Ma-Hock, L. (2015 (B)): the study duration is too short for a short-term repeated dose toxicity study. Only one dose group was examined which does not allow a dose-response related analysis. Test material was insufficiently characterised (chemical name, no purity or impurities). The number of animals per group (n= 3-5) is not sufficient to perform an appropriate statistical analysis.

Warheit, D.B. et al. (1996): the publication shows significant methodological deficiencies in the experimental set up and documentation. The experimental design is insufficiently documented. Frequency of exposure was not specified. Test material was insufficiently characterised (source, purity, impurities). Only male rats were tested and the number of animals per group was not given in publication. Furthermore, analytical parameters of the exposure atmosphere were measured (air flow, exposure concentration, and MMAD) were measured but not reported. Only selected parameters (cytotoxicity and pulmonary inflammatory responses (LDH and differential neutrophil levels in BAL fluid), alveolar macrophage functional parameters and histopathology of lung, trachea and heart) were investigated.

 

Conclusion - inhalation

The existing 28-day and 90-day inhalation studies with iron oxides in rats does not show any substance-related adverse effects. The effects of iron oxides after 28-day and 90-day inhalation is best compared with the effects seen with other poorly-soluble low-toxicity particles (PSLT), leading to a minimal or mild inflammatory response only at the maximum tolerated concentration in repeated dose toxicity studies via inhalation.

Based on the physico-chemical properties of the particles, direct systemic exposure to iron upon inhalation can be ruled out. The ingestion of particles cleared from the lungs can lead to a secondary exposure, the effects of which are sufficiently covered by oral studies and human data. Thus it can be concluded that the omission of “systemic” endpoints in the inhalation studies is not a serious shortcoming in the present context.

Based on the local effects particles can have in the respiratory tract and the physico-chemical properties of carbonyl iron, it is not expected that a longer duration would result in the detection of other effects, not solely determined by the PSP character of carbonyl iron. Thus it is not necessary to propose an additional inhalation study. It is concluded that carbonyl iron can be regulated as a PSP (inert dust) with no test-item specific toxicity.

In human epidemiological studies following prolonged inhalation exposure, no clinically significant adverse local or systemic effects were reported in humans.

 

 

 

Repeated dose toxicity – dermal:

There is no information available on repeated dermal toxicity. The conduct of repeated dose toxicity study is not considered to be required since inhalation of the substance is considered the most relevant route of human exposure. Furthermore, physicochemical and toxicological properties of the iron category substances do not suggest a significant rate of absorption through the skin (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006)

 

Repeated dose toxicity: other routes

Summary entry – Repeated dose toxicity: other routes

Another eleven references were also identified, representing in vivo experiments with investigations on repeated dose toxicity. These experiments were conducted with mice or rats receiving iron oxides (unspecified), (nano) diiron trioxide, (nano) triiron tetraoxide or superparamagnetic iron oxide via intraperitoneal, intralaryngeal or intratracheal administration. The study designs are not in accordance with accepted guidelines and are therefore of limited relevance for chemicals hazard assessment. The references usually lack significance due to, e.g., poor test item characterisation, low number of animals used, missing dose response relationship, unjustified dosing regime, or unphysiological route. It is therefore concluded that all references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The studies given below were included in the IUCLID for information purposes only:

Marshal, H.E. et al. (1987): the non-physiological route of administration via intralaryngeal instillation is not guideline conform and not suitable to assess repeated dose inhalation toxicity. Furthermore, the publication shows significant methodological deficiencies in the experimental set up and documentation. Only male rats and only one dose group were tested which does not allow a dose-response related analysis. Test material was administered only once a week. The number of animals per group was not specified. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Only selected parameters (epithelial mitotic rates for midtrachea, left intrapulmonary bronchus, and left lung bronchioles, and histopathological examination of respiratory tract) were investigated.

Feron, V.J. et al. (1972): the non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose inhalation toxicity. Furthermore, the publication shows significant methodological deficiencies in the experimental set up and documentation. Only one dose group were tested which does not allow a dose-response related analysis. Test material was administered only once a week. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Only selected parameters (weekly body weights, mortality, histopathology) were investigated.

Port, C.D. et al. (1973): the non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose inhalation toxicity. Furthermore, the publication shows significant methodological deficiencies in the experimental set up and documentation. Only one dose group were tested which does not allow a dose-response related analysis. A study duration of 3 weeks is too short, and the test material was administered only 3 times per week. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Only selected parameters (weekly histopathological examination of lungs and trachea) were investigated.

Shefner, A.M. et al. (1980): the non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose inhalation toxicity. Furthermore, the publication shows significant methodological deficiencies in the experimental set up and documentation. Only male animals and only one dose group were tested which does not allow a dose-response related analysis. Test material was administered only once a week. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Only selected parameters (mortality, weekly body weighing, histopathology of respiratory tract) were investigated.

Shefner, A.M. et al. (1982): the non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose inhalation toxicity. Furthermore, the publication shows significant methodological deficiencies in the experimental set up and documentation. Only males and only one dose group were tested which does not allow a dose-response related analysis. Test material was administered only once a week. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Only selected parameters (clinical signs, mortality, body weights, pathology, organ weights and histopathology) were investigated.

Liu, L. et al. (2004): abstract and results in tabular form are only in English language and the rest of the publication is in Chinese language. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess repeated dose toxicity. Furthermore, the modified test substance (Fe2O3 coated with glutamic acid) is not suitable for assessing the toxicity of Fe2O3.

Wright, J.L. et al. (1988): the non-physiological route of administration via intratracheal instillation is not guideline conform and not suitable to assess repeated dose inhalation toxicity. The publication shows significant methodological deficiencies in the experimental set up and documentation. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Frequency of exposure was not specified. Test material was insufficiently characterised (source, purity, impurities). Only females were tested. The number of animals per group (n=5) is not sufficient to perform an appropriate statistical analysis. Only selected parameters (pulmonary function tests and morphometric measurements) were investigated.

Katsnelson, B.A. et al. (2011A & B): the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess repeated dose toxicity. The self-synthesized test material was insufficiently characterised (no purity or impurities). Only females and only one dose group were tested which does not allow a dose-response related analysis. Test material was administered only 3 times a week. Study duration of 5 weeks is too short for a sub-chronic repeated dose toxicity study. Only selected parameters (body weights, exploratory behaviour, clinical biochemistry and histopathology of liver and spleen) were investigated.

Ma, P. et al. (2012): the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess repeated dose toxicity. The test material was insufficiently characterised (no purity or impurities). Only males were tested. Study duration of 1 week is too short for a short-term repeated dose toxicity study. The number of animals per group (n=5) is not sufficient to perform an appropriate statistical analysis. Only selected parameters (oxidative stress biomarkers & histopathology of liver and kidney) were investigated.

Ma, P. et al. (2011): abstract only in English language and the rest of the publication is in Chinese language. The non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess repeated dose toxicity. Study duration of 7 days is too short for a short-term repeated dose toxicity study. Only selected parameters (oxidative stress biomarkers (ROS, MDA and GSH) in lung tissue) were investigated.

Katsnelson, B.A. et al. (2011): the non-physiological route of administration via intraperitoneal injection is not guideline conform and not suitable to assess repeated dose toxicity. The self-synthesized test material has not been sufficiently characterised (no purity or impurities). Only females and only one dose group were tested which does not allow a dose-response related analysis. Test material was administered only 3 times a week. Study duration of 5 weeks is too short for a sub-chronic repeated dose toxicity study. Only selected parameters (body weights, exploratory behaviour, clinical biochemistry and histopathology of liver and spleen) were investigated.

Weissleder, R. et al. (1989): the non-physiological route of administration via injections is not guideline conform and not suitable to assess repeated dose toxicity. Publication shows significant methodological deficiencies in the experimental set up and documentation. The experimental design is insufficiently documented and not in accordance with any relevant guideline. Test material was insufficiently characterised (no information about the iron oxide form, purity or impurities). Type and site of injections, and the vehicle were not specified. The number of animals per group was not specified. Only two dose groups were tested which does not allow a dose-response related analysis. Study duration of 3 weeks is too short for a sub-acute repeated dose toxicity study.

 

 

 

 

Statement on the preferential use of human data in risk assessments for human health

 

(I) In almost 20 years of practical conduct of risk assessments under the “Existing Substances Regulation (793/93), human data has been given preference over animal studies. This is documented in the Technical Guidance Document in chapter 3.1 as follows: „Generally human data will only be available for existing substances. If both animal data and human data are available, as a general rule, well reported relevant human data for any given endpoint is to be given preference for the risk assessment.“ (ECB, 2003).

 

(II) Similarly, the US Environmental Protection Agency (EPA) in their guidance have stated that they look to human data whenever possible in completing human risk assessments: "If adequate human studies (confirmed for validity and applicability) exist, these studies are given first priority in the dose-response assessment, and animal toxicity studies are used as supportive evidence" (EPA, 1989). Often, such data can be obtained from epidemiological studies, which do not involve the intentional dosing of research participants, but rather evaluate the effects of exposures that have occurred in an occupational setting or because of the peculiarities of a specific geographical setting. Regardless of the origins of such human data, risk assessments based on human data have the advantage of avoiding the problems inherent in interspecies extrapolation" (EPA, 1993). In the same document, EPA also states: “The default assumptions that are of particular relevance to the issues raised by third-party intentional human dosing studies are those that bridge gaps between animal results and estimates of effects in humans. In the context of FIFRA, for example, EPA has routinely divided the calculated "safe" dose for animals by a factor of 10, to account for the possibility that humans are more sensitive to the substance being tested than are the animal species. Third-party submitters of human dosing studies have been particularly interested in modifying this default assumption by introducing data obtained directly from human studies.”

 

(III) When addressing the relevance and use of human data, ECHA guidance specifies the requirements for such studies as follows in section B.4.3.3 (human data) of their guidance, for the following four types of human data (ECHA, 2008):

 

Analytical epidemiology studies on exposed populations (case-control, cohort and cross-sectional studies) are useful for identifying a relationship between human exposure and effects and may provide the best data for risk assessment.

 

Descriptive or correlation epidemiology studies are useful for identifying areas for further research but are not very useful for risk assessment since they often can only identify patterns or trends but cannot ascertain the causal agent or degree of human exposure.

 

Case reports may demonstrate effects which cannot be observed in experimental animals. Thorough assessment of the reliability and relevance of case reports is needed because they often lack critical information on e.g. substance purity, human exposure, and effects.

 

Controlled studies in human volunteers are acceptable in very rare cases. Testing with human volunteers is strongly discouraged but when good quality data are already available, they should be used as appropriate in well justified cases.

 

In the case of cobalt and cobalt substances, the human studies that were used for the derivation of DNELs were assessed for their reliability and relevance, and were found to be of acceptable quality for the purpose envisaged.

 

(IV) Finally, the use of human data in risk assessment largely avoids a need for the application of assessment or extrapolation factors to account for differences in toxicokinetics, toxicodynamics, metabolic capacity and species sensitivity.

 

 

References

EPA (1989):        Risk Assessment Guidance for Superfund, Vol. 1: Human Evaluation Manual, EPA/540-1-89/002, US Environmental Protection Agency. available at: www.epa.gov/cgi-bin/claritgw?op-Display&document=clserv:OSWER:1175;&rank=4&template=epa

 

EPA (1993):        Reference Dose (RfD): Description and Use in Health Risk Assessments, § 1.3.2.2.1, US Environmental Protection Agency, background document, available at: www.epa.gov/IRIS/rfd.htm.

 

ECB (2003):        Technical Guidance Document on Risk Assessment in support of Commission Directive 93/67/EEC on Risk Assessment for new notified substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances, Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market, Part I, EUR 20418 EN/1

 

ECHA (2008)      Guidance on information requirements and chemical safety assessment, Guidance on information requirements and chemical safety assessment, Part B: Hazard Assessment, European Chemicals Agency, 2008

Justification for classification or non-classification

STOT-RE, oral

The classification criteria according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – repeated exposure, oral are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure to any of the members of the iron category and the no observed adverse effect level (NOAEL) via oral application is above the guidance value for a Category 1 classification of 10 mg/kg bw/day and above the guidance value for a Category 2 classification of 100 mg/kg bw/day. For the reasons presented above, no classification for specific target organ toxicant (STOT) – repeated exposure, oral is required.

 

Repeated dose toxicity, inhalation

The classification criteria according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – repeated exposure, inhalation are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after inhalation exposure to any of the members of the iron category when tested up to the maximum tolerated concentration. This is corroborated by an absence of adverse effects in humans. For the reasons presented above, no classification for specific target organ toxicant (STOT) – repeated exposure, oral is required.