Triiron bis(orthophosphate)

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

combined repeated dose and reproductive/developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-08-13 to 2001-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproductive/developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001-08-13 to 2001-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996-03-22

Deviations:

yes

Remarks:

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

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature, sealed under argon gas
- Stability under test conditions: test substance was stable throughout its period of use

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

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

Route of administration:

oral: gavage

Details on route of administration:

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

Vehicle:

other: water for injection

Details on oral exposure:

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

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

VEHICLE
- Lot no.: 0H93N

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

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

Duration of treatment / exposure:

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

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

equivalent to 6 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 20 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 60 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 201 mg Fe/kg bw/day

No. of animals per sex per dose:

12 males / 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

not applicable

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: No

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

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

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

WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

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

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

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

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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

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

Statistics:

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

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

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

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

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

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

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

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effect

Effect level:

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

Based on:

element

Remarks:

iron

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

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

Critical effects observed:

not specified

Conclusions:

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

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

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

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

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

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

7782-63-0

Molecular formula:

FeSO4 * 7H2O

Test material form:

solid

Details on test material:

- State of aggregation: blue-green crystalline powder
- Melting point: 64 °C
- Water-soluble

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

equivalent to 6 mg Fe/kg bw/day

Doses / concentrations 2

Dose / conc.:

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

Remarks:

equivalent to 20 mg Fe/kg bw/day

Doses / concentrations 3

Dose / conc.:

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

Remarks:

equivalent to 60 mg Fe/kg bw/day

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

4

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

5

Key result

Dose descriptor:

NOEL

Remarks:

reproduction

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

6

Key result

Dose descriptor:

NOEL

Remarks:

reproduction

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

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

1

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

2

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

3

Key result

Dose descriptor:

NOEL

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

4

Key result

Dose descriptor:

NOEL

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

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.