Iron sinter

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Description of key information

The evidence for the lack of toxicity to reproduction of the Iron, Mill scale, Iron ore agglomerates and Iron sinter category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. For comparison a reproduction/developmental toxicity screening test with a poorly soluble iron substance Fe3P was included. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (May 2022) attached to IUCLID section 13.2.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Vehicle:

other: 1% (w/v) aqueous methylcellulose

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks, or until evidence of mating was found
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): Individually

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

Details on study schedule:

- Age at mating of the mated animals in the study: Approx. 12 weeks

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 males and 10 females 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)".

Parental animals: Observations and examinations:

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: No

OTHER: 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.

Oestrous cyclicity (parental animals):

For 15 days before pairing, daily vaginal smears (dry) were taken from all Main phase females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the Main phase male, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight.
Seminal vesicles were subject to histopathological examination. 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.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Clinical signs, litter size, sex ratio, and bodyweight

GROSS EXAMINATION OF DEAD PUPS:
For offspring surviving to scheduled termination, a careful external examination for gross abnormalities was performed. Offspring that appeared normal externally were discarded without internal examination. Offspring which were externally abnormal were subjected to a full internal microscopic examination. Missing offspring and those grossly autolysed or cannibalised could not be examined; all other offspring which died before day 7 of age were examined as detailed. The necropsy also included an assessment for the presence of milk in the stomach, where possible.

Postmortem examinations (parental animals):

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)

Postmortem examinations (offspring):

Microscopic examination, as described in "Litter Observations", above.

Statistics:

please refer to "Any other information on material and methods incl. tables", below.

Reproductive indices:

- Percentage mating: Number animals mating / Animals paired x 100
- Conception rate (%): Number animals achieving pregnancy / Animals mated x 100
- Fertility index (%): Number animals achieving pregnancy / Animals paired x 100
- Gestation index (%): Number of live litters born / Number pregnant x 100
- Post - implantation survival index (%): Total number offspring born / Total number uterine implantation sites x 100

Offspring viability indices:

- Live birth index (%): Number live offspring on Day 1 after littering / Total number of offspring born x 100
- Viability index (%): Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering x 100
- Lactation index (%): Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering x 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Refer to detailed analysis of toxicological parameters in section 7.5.1.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles during the two week pre-pairing period showed that nearly all of the main phase females had regular 4 or 4/5 day oestrous cycles and it was considered that this parameter was not affected by treatment with Fe3P.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The pre-coital interval was unaffected by treatment, with animals mating at the earliest opportunity, when the female came into oestrus.
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.
The gestation length was within the normal range of 22 to 23 days for all animals and the gestation index was unaffected by treatment.

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

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

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
The mean number of implantations and the live litter size on Days 1, 4 and 7 were similar to control values and unaffected by treatment.
Offspring survival up to Day 7 of age was unaffected by parental treatment.

CLINICAL SIGNS (OFFSPRING)
Refer to table in "Any other information on results".

BODY WEIGHT (OFFSPRING)
Mean bodyweights of male and female offspring of all groups of parents treated with Fe3P were marginally lower than in Controls on Day 1 of age but differences did not attain statistical significance and there was considered to be no effect of treatment on mean bodyweight gains of the offspring between Days 1 and 4 of age. However, between Day 4 and Day 7 of age, mean bodyweight gains of male and female offspring of all groups of parents treated with Fe3P were lower than in Controls and the differences attained statistical significance in the 1000 or 300 mg/kg/day groups (77 - 80% of Controls) and they showed a dose response. Overall bodyweight gain, between Days 1-7, was statistically lower in all male offspring of all groups of parents treated with Fe3P.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring dying before scheduled termination or killed at scheduled termination on Day 7 of age did not reveal any findings that were attributed to parental treatment.

OTHER FINDINGS (OFFSPRING)
Sex ratio, as expressed in terms of % males, was not affected by parental treatment.

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Summary of offspring clinical observations

Observation	Group (Dose mg/kg/day)
	1 (0)	2 (100)	3 (300)	4 (1000)
	Number of offspring (litters) affected
Head: bruising and swelling	1(1)
Head: bruising		1(1)	1(1)	1(1)
Umbilical cord attached	1(1)
Missing	2(2)		1(1)	2(2)
Tail absent	1(1)
Dorsal surface: Scab and reddening		1(1)
Found dead		1(1)
Forepaw: Scab		1(1)
Colour: Pale			1(1)

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for Fe3P for reproductive and developmental effects was considered to be 1000 mg/kg/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined repeated dose toxicity study with the reproductive/ 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.

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

Justification for study design:

not applicable

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

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

distilled

Details on 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.

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female (same treatment group avoid sibling mating

Once mating was detected the, the respective males and females were separated. This day was designated day 0 postcoitum

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

Details on study schedule:

not specified

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

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight
Additional slides of the testes were prepared from the control- and high-dose group to examine the staging of spermatogenesis (not further specified).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not specified

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

- Number of live and dead pups on day 1 of lactation and daily thereafter
- Clinical signs determined at least once daily by visual inspection.
- Body weights of live pups determined on days 1 and 4 of lactation.
- Sex determined at days 1 and 4 of lactation,
- percentage of live males and females
- percent postnatal loss from days 0 to 4 of lactation.

Postmortem examinations (parental animals):

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).

Postmortem examinations (offspring):

SACRIFICE/ GROSS NECROPSY
- The F1 offspring surviving to 5 to 7 d postbirth were killed by decapitation. Pups were sexed and any external abnormalities recorded. The stomach was examined for the presence of milk. Where possible, defects or cause of death were determined or evaluated.

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.

Reproductive indices:

Mating index (%) = (females mated/females paired) × 100
Fertility index (%) = (pregnant females/females paired) × 100
Conception index (%) = (pregnant females/females mated) × 100
Gestation index (%) = (females with living pups on day 1/pregnant females) × 100
Duration of gestation

Offspring viability indices:

Viability index (%)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

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):

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

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

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

REPRODUCTIVE FUNCTION: SPERM MEASURES
1) Males
- 500, 1000 and 2000 mg/kg bw/d: normal testes weight and normal epididymites weights. Staging of spermatogenesis in the testes did not provide any evidence of test article-related impairment of the spermatogenetic cycle. The investigated parameters were not further specified.

REPRODUCTIVE PERFORMANCE
1) Males
- 500, 1000 and 2000 mg/kg bw/d: There were no treatment-related morphological findings in the reproductive organs, including the testes/epididymites, prostate gland, seminal vesicles preputial gland, ovaries, uterus, and vagina. There were no statistically or biologically significant changes in any of the parameters measured. mating index, fertility index, conception index, gestation index and duration of gestation

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproductive toxicity)

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

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

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

CLINICAL SIGNS
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: visual inspection of the F1 animals from days 0 to 4 of lactation were inconspicuous.

MORTALITY / VIABLITY
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: There were no statistically or biologically significant changes in any of the parameters measured. percentage of live F1 males and F1 females, percent postnatal loss from days 0 to 4 of lactation and viability index (F1) compared to the control.

GROSS PATHOLOGICAL FINDINGS
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d:
There was no effect of treatment on the results of the external macroscopic examination, like external abnormalities and presence of milk in the stomach.

OTHER EFFECTS
1) Males and Females
- 500, 1000 and 2000 mg/kg bw/d: the sex ratio of the offspring were normal.

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

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 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.,

However, 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.

Lastly, 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.

During the observation of the offspring animals (F1), no test item-related effects were observed for clinical signs, mortality, viability, bodyweight and weight changes, gross pathology and sex ratio.

The NOAEL for reproductive/developmental toxicity cannot be determined, based to the absence of adverse toxic effects in all investigated reproductive/developmental parameters. Based on the highest dose tested, a dose of FemTA 2000 mg/kg body weight/day (equivalent to Fe(III) 80 mg/kg bw/d) could be considered to cause no adverse reproductive/developmental effects.

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 considered to be 500 mg/kg bw/day for males and females. Furthermore, the NOAEL for developmental toxicity was determined to be 2000 mg/kg bw/day based on the absence of adverse toxic effects.

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 even beneficial to observe cumulative adverse effects of low magnitude influencing the female fertility and the entire spermatogenesis of male animals.

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 parental (P) 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.

In the offspring (F1) generation the suckling behaviour was not mentioned to be investigated. In most cases an alteration of the suckling behaviour should have had an impact of the body weight gain of the F1 animals. However, the body weight gain of the F1 treatment groups were normal compared to the F1 control group, indicating a normal suckling behaviour.

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 3

Endpoint:

screening for reproductive / developmental toxicity

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

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

Justification for study design:

not applicable

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

Vehicle:

other: water for injection

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: max. 14 days or until copulation was verified
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as gastation day 0

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

Details on study schedule:

not applicable

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

Parental animals: Observations and examinations:

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OBSERVATION OF DELIVERY:
- females that copulated were allowed to give birth naturally.
- delivery status was confirmed at the same time every day from gestation days 21 to 25.
- if delivery was completed by 10 am, that day was calculated as lactation day 0.

OBSERVATION OF NURSING
- dams were observed nursing every day until lactation day 4.

Oestrous cyclicity (parental animals):

The oestrus cycle of the females was observed once a day from the start of administration until the day copulation was verified. If oestrus was observed for two consecutive days, this was counted as one occassion.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight and epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
general condition (once a day), survival (once a day), body weight (day of birth and lactation day 4), number and sex of pups (at birth), number of stillbirths (at birth), number of live pups (at birth), and presence of external anomalies (at birth)

GROSS EXAMINATION OF DEAD PUPS: Yes, stillbirth and dead pups were fixed and stored for investigation.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: day after the final administration (dosing day 50)
- Maternal animals: day after the final administration (lactation day 6)
Any animals found to have died were necropsied promptly.

GROSS NECROPSY
- gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
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 (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.

Postmortem examinations (offspring):

SACRIFICE
Yes, surviving pups were sacrificed and then necropsied.

Statistics:

Significant difference between the control group and each administration group was tested and displayed as p<0.05 and p<0.01. The body weights of the pups were measured, and the mean and total values for each litter were calculated. The post-copulation general condition, body weights and food consumption of the females that did not conceive were excluded from the respective totals. The one animal of the 1000 mg/kg group that died during gestation was not used in the gestation index total.
Mean and standard deviation values for the following parameters were calculated for each group: body weight (parent animals, pups), food consumption, urine volume, urine specific gravity, haematology tests, blood biochemistry tests, absolute and relative organ weights, number of oestrus, number of days required for copulation, gestation period (day of delivery - day copulation verified), number of corpora lutea, number of implantations, total number of pups born (number of live pups born + number of stillbirths), number of live pups born, number of stillbirths, number of live pups on lactation day 4, and sex ratio (males/females). 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.
The copulation index, the conception index and the gestation index were tested using the χ2-test.
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.

Reproductive indices:

- implantation index: (number of implantations/number of corpora lutea) × 100
- delivery index: (total number of pups born/number of implantations) × 100
- birth index: (number of live pups on lactation day 0/number of implantations) × 100
- copulation index: (number of animals that copulated/number of animals co-housed) × 100
- conception index: (number of females that conceived/number of animals that copulated) × 100
- gestation index: (number of dams with live pups/number of females that conceived) × 100

Offspring viability indices:

- live birth index: (number of live pups on lactation day 0/total number of pups born) × 100
- viability index on lactation day 4: (number of live pups on lactation day 4/number of live pups on lactation day 0) × 100
- external anomalies index: (number of pubs with external anomalies/number of live pups born) × 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 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).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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
- 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.
- 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 in the 1000 mg/kg group. 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.

- 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.

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.

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.

REPRODUCTIVE FUNCTION: OESTRUS CYCLE
- 30, 100, 300 and 1000 mg/kg mg/kg groups. no significant difference in the number of oestrus in the pre-mating administration period (14 days) was observed between the control group and any administration group.

REPRODUCTION FUNCTION: SPERM MEASURES
- 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).

REPRODUCTIVE PERFORMANCE
- 30, 100, 300 and 1000 mg/kg mg/kg groups: there were no pairs that did not copulate in any group (copulation index: 100% in all groups). There was no significant difference in number of days for copulation between the control group and any administration group. There was no significant difference in conception index between the control group and any administration group.
There was no significant difference in gestation period between the control group and any administration group.
No dams in any group exhibited abnormal delivery.
The gestation index was 100% in all groups. The one dam (1000 mg/kg group) that died late in gestation was excluded from the gestation index total.
No dams in any group exhibited abnormal nursing.

- 100 mg/kg group: two females did not conceive (not significant).

- 30 and 300 mg/kg groups: number of corpora lutea, number of implantations and implantation index did not differ significantly from those in the control group.
-100 and 1000 mg/kg groups: numbers of implantations were significantly lower than in the control group, but there was no significant difference in the number of corpora lutea or the implantation index. The numbers of implantations in the 100 and 1000 mg/kg groups were within the test facility background data range (number of pregnant females: 159; number of corpora lutea: 14.7-16.7; number of implantations: 13.3-15.4; implantation index: 88.9-97.5%), and so this was not attributed to administration.

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 toxicity

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

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 201 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

iron

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

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

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL SIGNS
- 30, 100, 300, and 1000 mg/kg groups: observation of the external appearance of the pups revealed no anomalies in any group. There were no abnormalities in the general condition of the pups in any group.

MORTALITY / VIABILITY
- 30, 100, 300, and 1000 mg/kg groups: no significant difference between the control group and any administration group in the number of stillbirths, the number of pups on lactation day 0 or the live birth index. There was no significant difference between the control group and any administration group in the number of live pups on lactation day 4 or the viability on lactation day 4.

BODY WEIGHTS AND WEIGHT CHANGES
- 30, 100, 300, and 1000 mg/kg groups: no significant difference between the control group and any administration group in mean male and female pup body weights on lactation days 0 or 4, mean litter weights on lactation days 0 or 4, or total litter weights on lactation days 0 or 4.

GROSS PATHOLOGY
- 30, 100, 300, and 1000 mg/kg groups:
pups: no abnormalities observed in any group
stillbirth: no abnormalities observed in any group

OTHER EFFECTS
- 30, 100, 300, and 1000 mg/kg groups: no significant difference between the control group and any administration group in the total number of pups born, or the sex ratio on lactation day 0.
There was no significant difference between the control group and any administration group in the the sex ratio on lactation day 4 or the viability on lactation day 4.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 201 mg/kg bw/day

Based on:

element

Remarks:

iron

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

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.

With regard to the reproduction/development of the parent animals, no histopathological changes were observed in the testes, epididymis, seminal vesicles, prostate, ovaries, uterus, vagina or mammary glands at any dose level. Moreover, no changes due to administration were observed in the number of oestrus, copulation index, number of days required for copulation, conception index, gestation index, nursing, lactation, number of corpora lutea, number of implantations, implantation index or gestation period.

In the pups, no changes due to administration were observed in the total number of pups born, number of stillbirths, number of pups on lactation day 0, sex ratio on lactation day 0, delivery index, birth index, or live birth index. No changes due to administration were observed in the general condition of the pups. No changes due to administration were observed in the number of live pups on lactation day 4, sex ratio of the live pups on lactation day 4, or viability on lactation day 4. External observation revealed no changes due to administration. No changes due to administration were observed in the body weights. The necropsies of the pups revealed no changes due to administration.

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 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. With regard to the reproduction/development of the parent animals, a NOAEL for reproductive toxicity of 1000 mg/kg/day (equivalent to 201 mg Fe/kg bw/day) was concluded for the male and female rats due to the absence of any relevant toxicological effects. Also, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day (equivalent to 201 mg Fe/kg bw/day) was concluded for the offspring (F1 generation) based on the absence of any relevant toxicological effect.

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

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).

Justification for type of information:

see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996-03-22

GLP compliance:

yes

Limit test:

no

Justification for study design:

not applicable

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

Vehicle:

not specified

Details on exposure:

not specified

Details on mating procedure:

- Mating period: 14 days
- Proof of pregnancy: day after copulating, mating was verified by sperm in a vaginal rinse (day 0).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Note: duration of treatment includes a 2 week pre-mating exposure period
males: 42 days
females: 42 to 54 days

Frequency of treatment:

daily

Details on study schedule:

not specified

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

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight and epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
- body weight: measured on the day 0 and 4 at postpartum
- crown rump length (CRL): measured on the day 0 and 4 at postpartum
- sex ratio
- external findings in neonates
- litter size

Postmortem examinations (parental animals):

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

HISTOPATHOLOGY
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.

Postmortem examinations (offspring):

GROSS NECROPSY
External findings in neonates

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.

Reproductive indices:

- Mating rate (%) = (No. of female confiremd about coitus/ No. of male using mating) × 100
- Male fertility rate (%) = (No. of pregnant female/ No. of male using mating) × 100
- Female fertility rate (%) = (No. of pregnant female/ No. of female confirmed about coitus) × 100
- Parturition rate (%)
- Pre-implantation loss rate (%)
- Post-implantation loss rate (%)
- Mean gestation period (day) = period of pregnancy was calculated from mating day (day 0)
- Delivery day = period was estimated to identify the delivery (day 0)

Animal death before parturition was excluded in calculation of ‘Parturition rate’ and ‘Mean gestation period’

Offspring viability indices:

Birth rate and survival rate

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females
- 125, 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".

Dermal irritation (if dermal study):

not specified

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 compared to the control group.

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

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

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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".

REPRODUCTIVE FUNCTION. SPERM MEASURES
- 125, 250 and 500 mg/kg bw/day: no effects were stated for testis and epididymides weights.

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

REPRODUCTIVE PERFORMANCE
0, 125, 250 and 500 mg/kg bw/day: pre-implantation loss rates (%) were 14.4, 9.4, 14.3, and 9.8 at the 0, 125, 250, 500 mg/kg b.w./day treatment groups, respectively. Post-implantation loss rates (%) were 6.0, 6.0, 3.1, and 7.0. Furthermore, the mating rates (%) for the control group, 125, 250, 500 mg/kg b.w./day treatment groups were 93.3, 86.7, 100, and100, respectively. Fertility rates (%) were 73.3, 80.0, 93.3, and 73.3 for male rats, and 78.6, 92.3 , 93.3, and 73.3 for female rats. Parturition rates (%) were identical with the fertility rates for female animals, except for the high dose level (71.4 %; statistically significant different from control). Lastly, the period of pregnancy was 22.0, 22.1, 22.2, and 22.1 days for the control group, 125, 250, 500 mg/kg b.w./day treatment
groups, respectively.

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

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

water consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

water consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- 0, 125, 250 and 500 mg/kg bw/day: all data for survival rate were within the normal range and were as follows:
Postpartum day 0:
0 mg/kg bw/day: 96.8 %
125 mg/kg bw/day: 100 %
250 mg/kg bw/day: 98.0 %
500 mg/kg bw/day: 97.3 %

Postpartum day 0:
0 mg/kg bw/day: 97.4 %
125 mg/kg bw/day: 98.8 %
250 mg/kg bw/day: 98.5 %
500 mg/kg bw/day: 98.6 %

Body weight and weight changes:

no effects observed

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

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

BODY WEIGHT ND WEIGHT CHANGES
- 125, 250 and 500 mg/kg bw/day: no effect on body weight was observed.

Please also refer to the field "Attachedbackground material".

GROSS PATHOLOGICAL FINDINGS
- 125 mg/kg bw/day: crown rump length (CRL) of neonates were significantly decreased as compared with that of the control group for postpartum day 4 (p < 0.01). But the decrease did not correlate with body weights that is a main growth and developmental index. Since there was no dose-dependence of CRL decrease, it was concluded that the test substance did not influence the growth of neonates.

- 500 mg/kg bw/day: there was a case of acaudate at the high dose level.

Please also refer to the field "Attachedbackground material".

LITTER SIZE
- 0, 125, 250 and 500 mg/kg bw/day: all data for litter size were within the normal range.

Please also refer to the field "Attachedbackground material".

BIRTH RATE
- 0, 125, 250 and 500 mg/kg bw/day: all data for birth rate were within the normal range.

Please also refer to the field "Attachedbackground material".

SEX RATIO
- 0, 125, 250 and 500 mg/kg bw/day: all data for sex ratio were within the normal range.

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

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

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, neurobehaviour, reproductive function (testis and epididymis weight) and reproductive performance (pre-implantation lost, post-implantation loss and mating data). 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.

The investigation of the offspring revealed that survival rates were within the normal range and, therefore, no test item-related effects on survival rate was observed. In addition, no test item-related effects were observed on body weight, gross pathological findings, litter size, birth rate and sex ratio.

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. The NOAELs for reproductive toxicity and developmental toxicity cannot be determined due to the absence of adverse toxic effects up to a highest dose level of 500 mg/kg bw/day.

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, 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Introductory remark on read-across:

In this dossier, the endpoint repeated dose toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the Iron, Mill scale, Iron ore agglomerates and Iron sinter 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.

 

Toxicity to reproduction – animal data

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, reproductive function (testis and epididymis weight) and reproductive performance (pre-implantation lost, post-implantation loss and mating data). 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.

The investigation of the offspring revealed that survival rates were within the normal range and, therefore, no test item-related effects on survival rate was observed. In addition, no test item-related effects were observed on body weight, gross pathological findings, litter size, birth rate and sex ratio.

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 NOAELs for reproductive toxicity and developmental toxicity cannot be determined due to the absence of adverse toxic effects up to the highest dose of 500 mg/kg bw/day.

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. However, the reporting detail is insufficient so that the study was rated with reliability 4. Further details are reported in the IUCLID study record.

 

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 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 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 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.

With regard to the reproduction/development of the parent animals, no histopathological changes were observed in the testes, epididymis, seminal vesicles, prostate, ovaries, uterus, vagina or mammary glands at any dose level. Moreover, no changes due to administration were observed in the number of oestrus, copulation index, number of days required for copulation, conception index, gestation index, nursing, lactation, number of corpora lutea, number of implantations, implantation index or gestation period. In the pups, no changes due to administration were observed in the total number of pups born, number of stillbirths, number of pups on lactation day 0, sex ratio on lactation day 0, delivery index, birth index, or live birth index. No changes due to administration were observed in the general condition of the pups. No changes due to administration were observed in the number of live pups on lactation day 4, sex ratio of the live pups on lactation day 4, or viability on lactation day 4. External observation revealed no changes due to administration. No changes due to administration were observed in the body weights. The necropsies of the pups revealed no changes due to administration.

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. With regard to the reproduction/development of the parent animals, a NOAEL for reproductive toxicity of 1000 mg/kg/day (equivalent to 201 mg Fe/kg bw/day) was concluded for the male and female rats due to the absence of any relevant toxicological effects. Also, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day (equivalent to 201 mg Fe/kg bw/day) was concluded for the offspring (F1 generation) based on the absence of any relevant toxicological effect. The study is considered as reliable with restriction (RL2), details on the shortcomings are reported in the IUCLID study record.

 

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. During the observation of the offspring animals (F1), no test item-related effects were observed for clinical signs, mortality, viability, bodyweight and weight changes, gross pathology and sex ratio.

The NOAEL for reproductive/developmental toxicity cannot be determined, due to an absence of adverse toxic effects in all investigated reproductive/developmental parameters. Thus, 2000 mg/kg body weight/day (equivalent to Fe(III) 80 mg/kg bw/d) is the no adverse effect level for reproductive/developmental effects. 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), details on the shortcomings are reported in the IUCLID study record.

 

Toxicity to reproduction – human data

The meta-analysis by Pena-Rosas et al. 2015a included 61 randomised and quasi-randomised controlled trials carried out since 1936 in 27 countries, mostly conducted during the last 20 years. The review included 27 trials from 15 countries, but only 21 trials (with 5490 women) contributed data to the review. All studies compared daily versus intermittent iron supplementation. The methodological quality of included studies was mixed and most had high levels of attrition. Of the 21 trials contributing data, three studies provided intermittent iron alone, 14 intermittent iron + folic acid and four intermittent iron plus multiple vitamins and minerals in comparison with the same composition of supplements provided in a daily regimen.

The objective of this meta-analysis was to assess the benefits and harms of intermittent supplementation with iron alone or in combination with folic acid or other vitamins and minerals to pregnant women on neonatal and pregnancy outcomes.

Preventive iron supplementation reduced maternal anaemia at term by 70% (risk ratio (RR) 0.30; 95% confidence interval (CI) 0.19 to 0.46, 14 trials, 2199 women, low quality evidence), iron-deficiency anaemia at term (RR 0.33; 95% CI 0.16 to 0.69, six trials, 1088 women), and iron deficiency at term by 57% (RR 0.43; 95% CI 0.27 to 0.66, seven trials, 1256 women, low quality evidence). There were no clear differences between groups for severe anaemia in the second or third trimester, or maternal infection during pregnancy (RR 0.22; 95% CI 0.01 to 3.20, nine trials, 2125 women, very low quality evidence; and, RR 1.21; 95% CI 0.33 to 4.46; one trial, 727 women, low quality evidence, respectively), or maternal mortality (RR 0.33; 95% CI 0.01 to 8.19, two trials, 12,560 women, very low quality evidence), or reporting of side effects (RR 1.29; 95% CI 0.83 to 2.02, 11 trials, 2423 women, very low quality evidence). Women receiving iron were on average more likely to have higher haemoglobin (Hb) concentrations at term and in the postpartum period but were at increased risk of Hb concentrations greater than 130 g/L during pregnancy, and at term.

Compared with controls, women taking iron supplements less frequently had low birthweight new-borns (8.4% versus 10.3%, average RR 0.84; 95% CI 0.69 to 1.03, 11 trials, 17,613 women, low quality evidence), and preterm babies (RR 0.93; 95% CI 0.84 to 1.03, 13 trials, 19,286 women, moderate quality evidence). They appeared to also deliver slightly heavier babies (mean difference (MD) 23.75; 95% CI -3.02 to 50.51, 15 trials, 18,590 women, moderate quality evidence). None of these results were statistically significant. There were no clear differences between groups for neonatal death (RR 0.91; 95% CI 0.71 to 1.18, four trials, 16,603 infants, low quality evidence), or congenital anomalies (RR 0.88, 95% CI 0.58 to 1.33, four trials, 14,636 infants, low quality evidence).

Findings of this meta-analysis suggest that intermittent regimens produced similar maternal and infant outcomes as daily supplementation but were associated with fewer side effects and reduced the risk of high levels of Hb in mid and late pregnancy, although the risk of mild anaemia near term was increased.

 

 

The meta-analysis by Pena-Rosas et al. 2015b included 61 randomised and quasi-randomised controlled trials carried out since 1936 in 27 countries, mostly conducted during the last 20 years. The review included 27 trials from 15 countries, but only 21 trials (with 5490 women) contributed data to the review. All studies compared daily versus intermittent iron supplementation. The methodological quality of included studies was mixed and most had high levels of attrition. Of the 21 trials contributing data, three studies provided intermittent iron alone, 14 intermittent iron + folic acid and four intermittent iron plus multiple vitamins and minerals in comparison with the same composition of supplements provided in a daily regimen.

The objective of this meta-analysis was to assess the effects of daily oral iron supplements for pregnant women, either alone or in conjunction with folic acid, or with other vitamins and minerals as a public health intervention in antenatal care.

Preventive iron supplementation reduced maternal anaemia at term by 70% (risk ratio (RR) 0.30; 95% confidence interval (CI) 0.19 to 0.46, 14 trials, 2199 women, low quality evidence), iron-deficiency anaemia at term (RR 0.33; 95% CI 0.16 to 0.69, six trials, 1088 women), and iron deficiency at term by 57% (RR 0.43; 95% CI 0.27 to 0.66, seven trials, 1256 women, low quality evidence). There were no clear differences between groups for severe anaemia in the second or third trimester, or maternal infection during pregnancy (RR 0.22; 95% CI 0.01 to 3.20, nine trials, 2125 women, very low quality evidence; and, RR 1.21; 95% CI 0.33 to 4.46; one trial, 727 women, low quality evidence, respectively), or maternal mortality (RR 0.33; 95% CI 0.01 to 8.19, two trials, 12,560 women, very low quality evidence), or reporting of side effects (RR 1.29; 95% CI 0.83 to 2.02, 11 trials, 2423 women, very low quality evidence). Women receiving iron were on average more likely to have higher haemoglobin (Hb) concentrations at term and in the postpartum period, but were at increased risk of Hb concentrations greater than 130 g/L during pregnancy, and at term.

Compared with controls, women taking iron supplements less frequently had low birthweight newborns (8.4% versus 10.3%, average RR 0.84; 95% CI 0.69 to 1.03, 11 trials, 17,613 women, low quality evidence), and preterm babies (RR 0.93; 95% CI 0.84 to 1.03, 13 trials, 19,286 women, moderate quality evidence). They appeared to also deliver slightly heavier babies (mean difference (MD) 23.75; 95% CI -3.02 to 50.51, 15 trials, 18,590 women, moderate quality evidence). None of these results were statistically significant. There were no clear differences between groups for neonatal death (RR 0.91; 95% CI 0.71 to 1.18, four trials, 16,603 infants, low quality evidence), or congenital anomalies (RR 0.88, 95% CI 0.58 to 1.33, four trials, 14,636 infants, low quality evidence).

Findings of this meta-analysis suggest that daily iron supplementation reduces the risk of maternal anaemia and iron deficiency in pregnancy but the positive effect on other maternal and infant outcomes is less clear.

 

Toxicity to reproduction – Conclusion

The low order of toxicity to reproduction is manifested (i) in an absence of concern in screening studies in the rat for the soluble iron substances and (ii) an absence of any adverse effects on organs of reproduction in any repeated dose toxicity studies with the Iron, Mill scale, Iron ore agglomerates and Iron sinter category substances reported to date. Two large meta-analysis studies did not show any adverse effects in pregnancy outcome upon iron supplementation.

The evidence for the lack of toxicity to reproduction of the Iron, Mill scale, Iron ore agglomerates and Iron sinter category substances is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.

Effects on developmental toxicity

Description of key information

The evidence for the lack of toxicity to developmental of the Iron, Mill scale, Iron ore agglomerates and Iron sinter category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.2.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

other: human studies as described in IUCLID section 7.10

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Introductory remark on read-across:

In this dossier, the endpoint repeated dose toxicity is not addressed by substance-specific information, but instead by a weight of evidence approach based on collected information for all substances of the Iron, Mill scale, Iron ore agglomerates and Iron sinter 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.

 

Developmental toxicity – animal data

No higher-tier developmental toxicity study in animals is available for any member of the iron oxide category. However, there is sufficient weight of evidence from three high-quality guideline and GLP-compliant combined repeated dose toxicity study with the reproduction developmental toxicity screening test available. In all three studies highly soluble iron substances were administered to rats up to the highest dose of 2000 mg/kg bw/day. No signs of developmental toxicity were observed in any of the animals.

 

Developmental toxicity – human data

The meta-analysis by Haider et al. 2013 examined the exposure-response relation of dose of iron, duration of use, and haemoglobin concentration on birth weight and risk of low birth weight and preterm birth. Various comparisons were made evaluating the overall effect of iron, iron only, and iron with folic acid. In total 48 randomised trials (17,793 women) and 44 cohort studies (1,851,682 women) were included.

The randomised trials in this meta-analysis included studies in pregnant women of daily oral iron or iron and folic acid use compared with placebo, no iron, or no iron and folic acid. Trials of both supplementation and fortification were included. The analysis excluded trials of multiple vitamins and minerals, except those that examined the additional effect of iron or iron with folic acid in which all treatment groups received similar vitamins and minerals (except for iron or iron and folic acid). Trials examining maternal haematological, morbidity, and birth outcomes were included. Birth outcomes included mean duration of gestation (weeks), preterm birth (defined as birth of a neonate <37 weeks of gestation), mean birth weight (g), low birth weight (defined as birth weight <2500 g), mean birth length (cm), small for gestational age birth (defined as birth weight below the 10th centile of the gestational age and sex), stillbirth (defined as death of a foetus after 28 weeks of gestation), perinatal mortality (defined as deaths including stillbirths and neonatal deaths before 7 days of life), and neonatal mortality (defined as death of a neonate in the first month of life).

The observational studies in this meta-analysis included prospective cohort studies that allowed examination of the association of baseline anaemia with the above specified birth outcomes. Cross sectional and case-control studies were excluded, as these do not allow assessment of the temporal association between exposure and an outcome.

The meta-analysis showed that iron use increased maternal mean haemoglobin concentration by 4.59 (95% confidence interval 3.72 to 5.46) g/L compared with controls and significantly reduced the risk of anaemia (relative risk 0.50, 0.42 to 0.59), iron deficiency (0.59, 0.46 to 0.79), iron deficiency anaemia (0.40, 0.26 to 0.60), and low birth weight (0.81, 0.71 to 0.93). The effect of iron on preterm birth was not significant (relative risk 0.84, 0.68 to 1.03). Analysis of cohort studies showed a significantly higher risk of low birth weight (adjusted odds ratio 1.29, 1.09 to 1.53) and preterm birth (1.21, 1.13 to 1.30) with anaemia in the first or second trimester. Exposure-response analysis indicated that for every 10 mg increase in iron dose/day, up to 66 mg/day, the relative risk of maternal anaemia was 0.88 (0.84 to 0.92) (P for linear trend<0.001). Birth weight increased by 15.1 (6.0 to 24.2) g (P for linear trend=0.005) and risk of low birth weight decreased by 3% (relative risk 0.97, 0.95 to 0.98) for every 10 mg increase in dose/day (P for linear trend<0.001). Duration of use was not significantly associated with the outcomes after adjustment for dose. Furthermore, for each 1 g/L increase in mean haemoglobin, birth weight increased by 14.0 (6.8 to 21.8) g (P for linear trend=0.002); however, mean haemoglobin was not associated with the risk of low birth weight and preterm birth. No evidence of a significant effect on duration of gestation, small for gestational age births, and birth length was noted.

 

In a longitudinal study series on iron-deficiency anaemia and behaviour/cognition (Walter et al. 1998; Lozoff et al. 2012; Gahagan et al. 2019; East et al. 2020), infants were recruited from community clinics in low- to middle-income neighbourhoods in Santiago, Chile. Six-month-old infants were randomized to iron-fortified (12.7 mg/L) or low-iron (2.3 mg/L) formula for six months. The formula contained elemental iron as ferrous sulphate. The iron status (haemoglobin, mean cell volume, erythrocyte protoporphyrin and serum ferritin) of the infants was determined at 12 months of age. In addition, the iron status of the low-iron group was determined at 18 months of age. A follow-up study was conducted when the children were 10 years of age. During this follow-up study IQ, spatial memory, arithmetic achievement, visual-motor integration, visual perception, and motor functioning were measured. Furthermore. a covaried regression was used to compare iron-fortified and low-iron groups and considered haemoglobin level before randomization and sensitivity analyses to identify 6-month haemoglobin levels at which groups diverged in outcome. Another follow-up study was conducted at 16 years of age. At 16 years of age, cognitive ability, visual perceptual ability, visual memory and achievement in math, vocabulary, and comprehension were assessed, using standardized measures. Mean differences in developmental test scores were compared according to randomization group. Lastly, at age of 21 years, the young adults who took part in the trial during infancy were again investigated. The adults were assessed for neurocognition, emotion regulation, educational level, and attainment of adult developmental milestones.

In this study, low-iron formula was not significantly inferior to iron-fortified formula in the prevention of iron-deficiency anaemia. At 12 months of age, 3.8 % of the low-iron group had iron-deficiency anaemia compared to 2.8 % of the high-iron group (p = 0.35). However, a greater proportion of infants in the low-iron group was iron deficient at 12 months of age by the criterion of 2 of 3 measures of iron status in the deficient range, but mean differences in iron measures were minor from a clinical perspective. Infants in the low-iron group did not progress to iron-deficiency anaemia. All indicators of iron status improved in the low-iron group by 18 months of age. Infants in the high-iron group had higher 12-month haemoglobin and ferritin levels and mean cell volumes, and lower levels of erythrocyte protoporphyrin than those in the low-iron group (p < 0.005). This indicated that the amount of iron in the formula did affect overall iron status.

Children who received 12.7 mg/L of iron-fortified formula as infants had lower cognitive and visual-motor scores at 10 years than those receiving low-iron formula. However, the differences were only observed among children with the very highest or lowest haemoglobin levels on entry into the trial at 6 months. Children with high haemoglobin levels had lower 10-year test scores if they received iron-fortified formula, whereas those with low haemoglobin levels had higher scores. In conclusion, this study indicates poorer long-term developmental outcome in infants with high haemoglobin concentrations who received formula fortified with iron. Most infants showed no developmental effects of iron-fortified formula, and those with low haemoglobin levels in infancy had higher 10-year test scores if they received iron-fortified formula.

Adolescents who were randomized to iron-fortified formula (12 mg/L) between 6 and 12 months had lower cognitive scores, at 16 years, compared to those who had received low-iron formula (2.3 mg/L). The low-iron group performed better than the iron-fortified group on eight out of nine measures, with statistically significant differences in verbal comprehension, arithmetic achievement, and spatial memory. Moreover, there was no impact on these findings when infants who became iron deficiency anaemia (IDA) or were treated with oral iron therapy were excluded from the analyses. Furthermore, an interaction between 6-month haemoglobin status and iron supplementation at 16 years was found, but only for visual motor integration (VMI). Participants who had low 6-month haemoglobin had higher scores for VMI if they had been randomized to iron-fortified formula and those who had high 6-month haemoglobin had higher scores for VMI if they had been randomized to low-iron formula.

This study found that lower age 10 cognitive abilities stemming from consuming iron-fortified formula in infancy were associated with poorer neurocognitive functioning, emotional awareness, and educational attainment in young adulthood. Additionally, consumption of iron-fortified formula was marginally directly related to poorer verbal memory and slower mental processing in young adulthood, and modest dosage effects of iron-fortified formula were found for lower educational attainment and slower processing speed. In the current study, the children at age 10 tested broadly in the normal intelligence range, with those receiving iron-fortified formula scoring in the slightly lower normal range than those receiving low-iron supplementation. Individuals who received iron-fortified formula in infancy and had relatively low IQ and low spatial memory at age 10 had the poorest neurocognitive performance as young adults. Lastly, results of the regression analyses also showed that participants who received iron-fortified formula and had average above average age 10 IQ or spatial memory scored equally as well on neurocognitive tests as those or who received low-iron formula in infancy.

The longitudinal study, as described above, has experimental and reporting deficiencies. First of all, during the treatment period it was not recorded, if the infants received solid food besides the diet formula and breast feeding and what kind of solid food they were given to eat. Therefore, it cannot be ruled out that the infants also received iron from other food source and the overall amount of iron consumed by the infants might have been higher or lower as assumed. In addition, as stated by East et al. (2020), differences in performance between the iron-fortified and low-iron groups could be the results of an interaction between the level of iron and another formula component. Furthermore, as pointed out by Gahagan et al. (2019), the iron absorption was not measured and the exact amount of iron metabolised is unknown.

In this longitudinal study, haemoglobin levels were measured in infants in order to determine the iron status, however, there also are other measures of iron status that could be investigated to obtain more precise measurement of iron. For example, serum iron could be determined, since not all iron will be bound by haemoglobin. Furthermore, as pointed out by Gahagan et al. (2019), venous measurements of haemoglobin would have been more accurate as capillary haemoglobin measurement. Since infants were only investigated by venous measurements when the capillary measurements indicated a problem with the haemoglobin level, it might be that infants were included in the study which would have been excluded otherwise, if venous measurements would have been used.

Confounding factors such as smoking by the mothers or other household members were not controlled for in the study. Also, the possibility of alcohol consumption by the mothers during pregnancy was not determined. Such factors might also have affect the developmental outcome. In addition, the authors did not determine the iron status prenatally or neonatally, as mentioned by Gahagan et al. (2019). Therefore, it could be that the iron status at that time might also play a role in the outcome of the study. Although adjusted estimates from randomised trails and cohort studies were used, these data were of observational nature. These associations still could have been biased owing to residual confounding, in either direction, depending on the nature of the residual confounding. Associations with several outcomes could not be evaluated owing to the paucity of data (stillbirths, neonatal and perinatal mortality in iron use meta-analyses; birth length, neonatal mortality in the cohort studies analysis). A small number of trials had evaluated the effect of iron fortification in pregnant women, so a separate meta-analysis for fortification trials could not be done. Significant heterogeneity existed for several outcomes that could not be explained substantially by pre-specified subgroups. This limits the understanding of the association in various settings and restricts the generalisability of the findings. For the exposure-response analysis of cohort studies, mean haemoglobin concentrations for most studies were missing. An assumption was made, that countries within the same category (low-, middle-, or high-income) would have similar mean haemoglobin concentrations. These mean values were used for analysis, which may have introduced bias towards the null due to random measurement error.

 

Developmental toxicity – Conclusion

The low order of developmental toxicity is manifested (i) in an absence of concern in screening studies in the rat for the soluble iron substances and (ii) an absence of any adverse effects on organs of reproduction in any repeated dose toxicity studies with the Iron, Mill scale, Iron ore agglomerates and Iron sinter category. A large meta-analysis study did not show any adverse effects in the development during pregnancy and post-partum upon iron supplementation.

The evidence for the lack of developmental toxicity of the iron oxides category is taken from read-across studies with soluble iron substances with a higher bioaccessibility and bioavailability, which constitutes an intrinsic conservatism. Details on the read-across approach are given in the report generated in accordance with the ECHA Read-across Assessment Framework (March 2017) attached to IUCLID section 13.