Diiron trioxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for 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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

publication

Title:

Ferrous sulfate heptahydrate

Author:

MHLW

Year:

2003

Bibliographic source:

Toxicity Testing Reports of Environmental Chemicals, vol 10, 2003

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)

GLP compliance:

yes

Limit test:

no

Justification for study design:

not applicable

Test material

Test material information

Constituent 1

Reference substance name:

Ferrous sulphate heptahydrate

Cas Number:

7782-63-0

IUPAC Name:

Ferrous sulphate heptahydrate

Test material form:

solid

Details on test material:

- Analytical purity (batch A): 91.1 wt%
- Analytical purity (batch B): 90.6 wt%
- Impurities (batch A): Mg 0.30 %, Mn 0.19 %, Zn 75 ppm
- Impurities (batch B): Mg 0.28 %, Mn 0.16 %, Zn 84 ppm

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
- Weight at start of administration: males: 341 - 383 g; females: 222 - 255 g

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

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.

Testing facility: Hashima Laboratory, Nihon Bioresearch Inc.

Duration of treatment / exposure:

males: 49 days
females: 14 days before mating to day 5 of lactation

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

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 ¿2-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, ¿-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: ¿-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

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

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:

With regard to the reproduction/development of the parent animals, no changes attributable to the test substance were noted in terms of the number of estrous cases, copulation index, number of days before copulation, fertility index, gestation length, gestation index, delivery conditions, nursing conditions, number of corpora lutea, number of implantation sites, or implantation rate.

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, 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.

Regarding 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.