Triiron bis(orthophosphate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Triiron bis(orthophosphate) does not show adverse effects towards male and female reproductive organs in oral repeated dose toxicity studies in animals. Extensive information on an absence of severe adverse effects in humans is available and discussed below.

Link to relevant study records

Reference

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

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

Justification for study design:

not applicable

Specific details on test material used for the study:

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

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

Route of administration:

oral: gavage

Vehicle:

other: water for injection

Details on exposure:

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

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

VEHICLE
- Lot no.: 0H93N

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

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

Duration of treatment / exposure:

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

Frequency of treatment:

daily

Details on study schedule:

not applicable

Dose / conc.:

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

Remarks:

equivalent to 6 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 20 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 60 mg Fe/kg bw/day

Dose / conc.:

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

Remarks:

equivalent to 201 mg Fe/kg bw/day

No. of animals per sex per dose:

12 males / 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

not applicable

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: No

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

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

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

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

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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

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

Postmortem examinations (parental animals):

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

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The weights of the brain (cerebrum, cerebellum, medulla), pituitary, thyroid, thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymis, ovaries and uterus were measured. The relative organ weights of all organs weighed were calculated. The lungs, pituitary, thyroid, trachea, pancreas, salivary glands (sublingual and submandibular), oesophagus, stomach, duodenum, coelenteron, ileum, caecum, colon, rectum, lymph nodes (submandibular, mesenteric), bladder, seminal vesicles, testes, edpididymis, eyeballs, prostate, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, harderian gland, bone marrow (sternal, femoral), bones (sternum, femur) and mammary glands (females only) were fixed.
Twenty five days after mating, the females that did not give birth were exsanguinated under ether anaesthesia and necropsied. The number of corpora lutea and the number of implantations were counted. The brain (cerebrum, cerebellum, medulla), pituitary, thyroid, thymus, heart, liver, spleen, kidneys, adrenals, ovaries, lungs, trachea, pancreas, salivary glands (sublingual and submandibular), oesophagus, eyeballs, stomach, duodenum, coelenteron, ileum, caecum, colon, rectum, lymph nodes (submandibular, mesenteric), bladder, uterus, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, harderian gland, bone marrow (sternal, femoral), bones (sternum, femur) and mammary glands were fixed.

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

Postmortem examinations (offspring):

SACRIFICE
Yes, surviving pups were sacrificed and then necropsied.

Statistics:

Significant difference between the control group and each administration group was tested and displayed as p<0.05 and p<0.01. The body weights of the pups were measured, and the mean and total values for each litter were calculated. The post-copulation general condition, body weights and food consumption of the females that did not conceive were excluded from the respective totals. The one animal of the 1000 mg/kg group that died during gestation was not used in the gestation index total.
Mean and standard deviation values for the following parameters were calculated for each group: body weight (parent animals, pups), food consumption, urine volume, urine specific gravity, haematology tests, blood biochemistry tests, absolute and relative organ weights, number of oestrus, number of days required for copulation, gestation period (day of delivery - day copulation verified), number of corpora lutea, number of implantations, total number of pups born (number of live pups born + number of stillbirths), number of live pups born, number of stillbirths, number of live pups on lactation day 4, and sex ratio (males/females). Bartlett's test for homogeneity of variance was performed, and if there was homogeneity of variance, Dunnett's test was performed. If no homogeneity of variance was found, a Dunnett-type rank test was performed.
The copulation index, the conception index and the gestation index were tested using the χ2-test.
For tissue where effects indicative of toxicity were observed in the 1000 mg/kg group, the histopathological findings for the other dose groups were compared with the control group and between groups using the Dunnett-type rank test.

Reproductive indices:

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

Offspring viability indices:

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

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

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

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 1000 mg/kg group: urine volume was significantly higher and the urine specific gravity was significantly lower than in the control group (p<0.01).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

HISTOPATHOLOGICAL FINDINGS. NON-NEOPLASTIC
1) Males (surviving males):
1000 mg/kg group:
- thymus: very mild atrophy was observed in two males.
- stomach: moderate ulceration of the glandular stomach in one male, very mild erosion of the glandular stomach in one male, mild or moderate inflammatory cell infiltration of the glandular stomach submucosa in two males, mild haemorrhage of the glandular stomach submucosa in one male, and very mild vacuolisation of the forestomach epithelium in one male.
- liver: yellow-brown pigment deposition in the periportal hepatocytes (significant difference compared to control) in all six males, and yellow-brown pigment deposition in the periportal Kupffer cells in three males. The yellow-brown pigment deposition was very mild or mild. On special staining of the livers, bile staining afforded no staining, iron staining afforded mild or moderate periportal staining, and wear-and-tear pigment staining afforded very mild or mild periportal staining.
- spleen: extramedullary haematopoiesis in four males, and yellow-brown pigment deposition in the red pulp in all six males. The extramedullary haematopoiesis observed was very mild to moderate. The yellow-brown pigment deposition in the red pulp was mild or moderate in the 1000 mg/kg group. It should be noted that the yellow-brown pigment deposition observed in the red pulp constituted a significant difference compared to the control group. For the extramedullary haematopoiesis observed in the 1000 mg/kg groups, the number of males affected and the severity differed compared to the control group.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

REPRODUCTION FUNCTION: SPERM MEASURES
- 300 mg/kg group: absolute testis weights were significantly higher than in the control group (no significant difference was observed in the 1000 mg/kg group; not test item-related finding).

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

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

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

element

Remarks:

iron

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

element

Remarks:

iron

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

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

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

Critical effects observed:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

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

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

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

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

201 mg/kg bw/day

Based on:

element

Remarks:

iron

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

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

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Introduction to read-across approach

During the literature search and data gap analysis it became obvious that the overall database on substance-specific human health hazard data for triiron bis(orthophosphate) is too scant to cover all REACH endpoints. Therefore, the remaining data gaps had to be covered by either experimental testing or read-across from similar substances.

Selected endpoints for the human health hazard assessment are addressed by read-across, using a combination of data on the phosphate moiety and the iron moiety (or one of its readily soluble salts). This way forward is acceptable, since triiron bis(orthophosphate) dissociates to the phosphate anion and the iron cation upon dissolution in aqueous media.

Iron exists in three stable oxidation states (i.e. 0, +2 and +3). As to the speciation of iron under physiological conditions, Fe2+must be assumed to be the prevailing species under mildly acidic conditions according to the Pourbaix diagram for iron:

 

In a detailed review (Kraemer, 2004) it is described in detail that siderophores, i.e. organic ligands with a specific affinity for iron together with pH have a strong influence on dissolution of iron substances. The thermodynamically stable form of iron under environmentally and most physiologically relevant conditions is the trivalent Fe3+ cation. The transformation rate of dissolved Fe2+ to the stable Fe3+ at neutral pH is rapid (within minutes); whereas at acidic pH(<4), Fe2+ will be the more stable valence state. Upon dissolution, the speciation is dependent on pH and redox potential of the environment. More specifically, trivalent iron oxides will release Fe3+ ions upon the limited dissolution during the GI tract passage, and will remain in this state. Conversely, any divalent iron oxide will initially release Fe2+ ions, which however are only stable in acidic gastric medium; upon entry into the slightly alkaline intestinal compartment, rapid conversion to Fe3+ ions must be assumed.

 

Once the constituents of triiron bis(orthophosphate) become bioavailable (i.e. in the acidic environment in the gastric passage or after phagocytosis by pulmonary macrophages), the overall toxicity of the dissociated substance can be described by the toxicity of the individual constituents. Since synergistic effects are not expected, the human health hazard assessment of the assessment entity triiron bis(orthophosphate) consists of an individual assessment of the assessment entities iron cation and the phosphate anion. The iron cation and the phosphate anion are considered to represent the overall toxicity of triiron bis(orthophosphate) in a manner proportionate to the phosphate and the metal (represented by one of its readily soluble salts). Based on the above information, unrestricted read-across is considered feasible and justified.

The hazard information of the individual constituents was obtained from publicly available peer-reviewed risk assessment documents, such as EFSA opinions, WHO recommendations for human nutrition.

 

Iron

Animal data

In a repeated dose toxicity study with reproductive and developmental screening (according to OECD 422 and under GLP), iron(II)sulfate was administered to rats at doses of 30, 100, 300 and 1000 mg/kg bw/day via gavage (Pharmaceutical and Food Safety Bureau 2002).

General observation revealed salivation in males and females in the ≥300 mg/kg bw/day 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.

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 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.

 

Human data

There is no evidence of any reprotoxic for (non-haem) iron compounds. Iron is abundantly available in the environment and in food, and is extensively distributed throughout the human body, which is contradictory to allegation of reprotoxic potential.

According to the European Food Safety Authority the EU population the reference intake is about 18–72 mg iron per day for the general population. During pregnancy, 450 mg Fe is needed to allow increased erythropoiesis, while 270-300 mg and 50-90 mg are transferred to the foetus and placenta, which gives a total extra demand of 770-840 mg. This demand corresponds to approx. 3 mg Fe/day and will be provided by an intake of 30 mg Fe/day and is the rationale for the recommended higher iron intake in pregnancy (FNB, 2001; Arbeitsgruppe “Referenzwerte für Nährstoffzufuhr, 2000).

Three main factors that affect iron balance are absorption (intake and bioavailability of iron), losses, and stored amount. The interrelationship among these factors has recently been described in mathematical terms, making it possible to predict, for example, the amount of stored iron when iron losses and bioavailability of dietary iron are known (Hallberg L et al., 1998). In states of increased iron requirement or decreased bioavailability, the regulatory capacity to prevent iron deficiency is limited (Hallberg L et al., 1995). However, the regulatory capacity seems to be extremely good in preventing iron overload in a state of increased dietary iron intake or bioavailability (Hallberg L et al., 1998).

Reprotoxic potential of the abundantly available essential element iron are grossly implausible and can be safely excluded. Therefore, further testing should not be considered, inter alia for reasons of animal welfare. In conclusion, conduct of studies for reproductive toxicity on iron is considered to be scientifically unjustified (in accordance with regulation (EC) 1907/2006, Annex XI, Section 1.1.3 and 1.2).

 

Phosphate

A registration dossier shall contain information on the human health hazard assessment (regulation 1907/2006, Art.10). However, it is considered that the information requirements for orthophosphate as laid down in annex VII to IX can be fulfilled by adaptation of the standard testing regime according to Annex XI, points 1.1.3, 1.2, as presented in the following:

Phosphorus is most commonly found as the phosphate ion, with phosphorus in its pentavalent form. Thus, in the following the term phosphorus refers to phosphate, including its major form orthophosphate.

(1) A large part of human nutrition consists of phosphorus as cited by EFSA, 2015:

The major dietary contributors to phosphorus intake are foods high in protein content, i.e. milk and milk products followed by meat, poultry and fish, grain products and legumes. Based on data from 13 dietary surveys in nine European Union countries, mean phosphorus intakes range from 265 to 531 mg/day in infants, from 641 to 973 mg/day in children aged 1 to < 3 years, from 750 to 1202 mg/day in children aged 3 to < 10 years, from 990 to 1601 mg/day in children aged 10 to < 18 years and from 1000 to 1767 mg/day in adults (≥18 years).(EFSA, 2015)

 

(2) The EFSA and JECFA concluded on phosphorus:

The available data[derived from short and long term studies in rodents and humans, and summarised by EFSA, 2005]indicate that normal healthy individuals can tolerate phosphorus intakes up to at least 3000 mg phosphorus per day without adverse systemic effects. In some individuals, however, mild gastrointestinal symptoms, such as osmotic diarrhoea, nausea and vomiting, have been reported if exposed to supplemental intakes >750 mg phosphorus per day. Estimates of current intakes of phosphorus in European countries indicate total mean dietary and supplemental intakes around 1000-1500 mg phosphorus per day, with high (97.5 percentile) intakes up to around 2600 mg phosphorus per day. There is no evidence of adverse effects associated with the current intakes of phosphorus.(EFSA, 2005)

Long-term effects of dietary phosphoric acid in three generations of rats have been investigated (Bonting and Jansen, 1956). The animals received diets containing 1.4% and 0.75% phosphoric acid (equivalent to approximately 200 and 375 mg phosphorus/kg body weight/day) for 90 weeks. No harmful effects on growth or reproduction were observed, and also no significant differences were noted in haematological parameters in comparison with control animals. There was no acidosis, nor any change in calcium metabolism. The quality of these older studies would be considered limited by current standards. (EFSA, 2005)

JECFA reviewed the available data from studies in mice and rats and concluded that dosing with phosphoric acid and inorganic phosphate salts does not induce maternal toxicity or teratogenic effects. Maximum dose levels tested for the various inorganic phosphate salts varied between 130 and 410 mg phosphorus/kg bodyweight (JECFA, 1982).(EFSA, 2005)

(3) Exposure of breast-fed babies to phosphorus via mother milk: A breast-fed of 3 months age has an average weight of 6.5 kg (WHO 2013) and the infant ingests approx. 180mL/kg bw of milk per day (Riordan 2001), being 1170 mL for a baby at an age of 3 months. The phosphorus content of mother milk is approx. 140 mg/mL at 90 days post partum (Atkinson et al. 1995; EFSA, 2015). This results in a total “exposure” for a 3 month old baby of 163.8 mg phosphorus per day, being 25.2 mg/kg bw/day.

A detailed evaluation of the underlying single studies is not provided here, in order to avoid unnecessary duplication of the work already performed by an EU-nominated expert body. Based on the findings evaluated in the EFSA document, an upper intake level (UL) cannot be established based on the effect of a high phosphorus intake on the activity of calcium regulating hormones, which the expert body considers not to be adverse in themselves, and which have no demonstrable effects on bone mineral density and skeletal mass. (EFSA, 2005)

 

Based on the above given arguments, one may safely assume that human exposure towards phosphorus substances exerts any adverse effects of toxicological relevance after chronic exposure.

In conclusion, the conduct of any further toxicity studies with chronic exposure in animals would not contribute any new information and is therefore not considered to be required.

Triiron bis(orthophosphate)

Since no toxicity study on fertility impairment is available specifically for Triiron bis(orthophosphate), information on the individual constituents iron and phosphate will be used for the hazard assessment and when applicable for the risk characterisation of triiron bis(orthophosphate). For the purpose of hazard assessment of triiron bis(orthophosphate), no hazard was identified for the individual assessment entities iron and phosphate. Consequently, no hazard is identified for the assessment entity triiron bis(orthophosphate) with regard to impairment of fertility.

References:

 

Appel, M.J. et al. (2001). Disposition, accumulation and toxicity of iron fed as iron (II) sulfate or as sodium iron EDTA in rats. Food and Chemical Toxicology 39: 261 - 269.

Atkinson S, Alston-Mills B, Lonnerdal B and Neville MC (1995): B. Major minerals and ionic constituents of human and bovine milks. In: Handbook of Milk Composition. Ed Jensen RJ. Academic Press, California, USA, 593-619.

Bonting SL and Jansen B C (1956): The effect of a prolonged intake of phosphoric acid and citric acid in rats. Voeding 17: 137.

Brock C, Curry H, Hama C, Knipfer M, Taylor L (1985). Adverse effects of iron supplementation: A comparative trial of wax-matrix iron preparation and conventional ferrous sulfate tablets. Clin Ther 7: 568-573.

Cook JD, Carriaga M, Kahn SG, Schack W, Skikne BS (1990). Gastric delivery system for iron supplementation. Lancet 336: 1136-1139.

Coplin M, Schuette S, Leichtmann G, Lasher B (1991). Tolerability of iron: A comparison of bis-glycino iron(II) and ferrous sulfate. Clin Ther 13: 606-612.

EFSA (2005): Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the Tolerable Upper Intake Level of Phosphorus, The EFSA Journal, 233, 1-19.

EFSA (2015): Scientific Opinion on Dietary Reference Values for phosphorus, EFSA Panel on Dietetic Products, Nutrition and Allergies. The EFSA Journal, 13(7): 4185.

Frykman E, Bystrom M, Jansson U, Edberg A, Hansen T (1994). Side effects of iron supplements in blood donors: Superior tolerance of heme iron. J Lab Clin Med 123: 561-564.

Green R, Charlton R, Seftel H, Bothwell TH, Maget F (1968). Body iron excretion in man. A collaborative study. Am J Med 45: 336-353.

Hallberg L and Rossander L (1984). Improvement of iron nutrition in developing countries: comparison of adding meat, soy protein, ascorbic acid, citric acid, and ferrous sulphate on iron absorption from a simple Latin American-type of meal. Am J Clin Nutr 39: 577-583.

Hallberg L et al. Iron balance in menstruating women. European Journal of Clinical Nutrition, 1995, 49:200–207.

Hallberg L, Hulthén L, Garby L. Iron stores in man in relation to diet and iron requirements. European Journal of Clinical Nutrition, 1998, 52:623–631.

Hunt JR and Roughead ZK (2000). Adaptation of iron absorption in men consuming diets with high or low iron bioavailability. Am J Clin Nutr 71: 94-102.

JECFA (Joint FAO/WHO Expert Committee on Food Additives) (1982): Toxicological evaluations of certain food additives. Twenty-sixth report of the joint FAO/WHO Expert Committee on Food Additives. WHO Additives Series No 17.

Mulvihill B, Kirwan FM, Morrissey PA, Flynn A (1998). Effects of myofibrillar muscle protein on the in vitro bioavailability of non-haem iron. Int J Food Sci Nutr 49: 187-192.

Nutrient and energy intakes for the European Community: a report of the Scientific Committee for Food. Brussels (1993), Commission of the European Communities.

Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Tokyo (2002). Combined repeated dose and reproductive/developmental toxicity study of iron II sulfate heptahydrate oral administration in rats. Hashima Laboratory, Nihon Bioresearch Inc., 6-104 Majima, Fukuju-cho, Hashima, Gifu. Report No. 100520

Reddaiah VP, Prasanna P, Ramachandran K, Nath LM, Sood SU, Madan N, Rusia U (1989). Supplementary iron dose in pregnant anemia prophylaxis. Ind J Pediatr 65: 109-114.

Rossander L (1987). Effect of Fiber on iron absorption in man. Scand J Gastroenterol 22 (Suppl) 129: 68-72

Rossander-Hulthén L, Hallberg L (1996). Prevalence of iron deficiency in adolescents. In: Hallberg L, Asp N-G, eds. Iron nutrition in health and disease. London, John Libby, 149–156.

Sato, H. et al. (1985). Oral subchronic toxicity studies of ferric chloride in F344 rats. Bull. Natl. Inst. Hyg. Sci. (103): 21 - 28.

WHO (2013): The WHO Child Growth Standards. Available at: http://www.who.int/childgrowth/standards/en/index. html.

Effects on developmental toxicity

Description of key information

Triiron bis(orthophosphate) does not show adverse effects towards male and female reproductive organs in oral repeated dose toxicity studies in animals. Extensive information on an absence of severe adverse effects in humans is available and discussed below.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Introduction to read-across approach

 

During the literature search and data gap analysis it became obvious that the overall database on substance-specific human health hazard data for triiron bis(orthophosphate) is too scant to cover all REACH endpoints. Therefore, the remaining data gaps had to be covered by either experimental testing or read-across from similar substances.

 

Selected endpoints for the human health hazard assessment are addressed by read-across, using a combination of data on the phosphate moiety and the iron moiety (or one of its readily soluble salts). This way forward is acceptable, since triiron bis(orthophosphate) dissociates to the phosphate anion and the iron cation upon dissolution in aqueous media.

Iron exists in three stable oxidation states (i.e. 0, +2 and +3). As to the speciation of iron under physiological conditions, Fe2+must be assumed to be the prevailing species under mildly acidic conditions according to the Pourbaix diagram for iron:

 

In a detailed review (Kraemer, 2004) it is described in detail that siderophores, i.e. organic ligands with a specific affinity for iron together with pH have a strong influence on dissolution of iron substances. The thermodynamically stable form of iron under environmentally and most physiologically relevant conditions is the trivalent Fe3+ cation. The transformation rate of dissolved Fe2+ to the stable Fe3+ at neutral pH is rapid (within minutes); whereas at acidic pH(<4), Fe2+ will be the more stable valence state. Upon dissolution, the speciation is dependent on pH and redox potential of the environment. More specifically, trivalent iron oxides will release Fe3+ ions upon the limited dissolution during the GI tract passage, and will remain in this state. Conversely, any divalent iron oxide will initially release Fe2+ ions, which however are only stable in acidic gastric medium; upon entry into the slightly alkaline intestinal compartment, rapid conversion to Fe3+ ions must be assumed.

 

Once the constituents of triiron bis(orthophosphate) become bioavailable (i.e. in the acidic environment in the gastric passage or after phagocytosis by pulmonary macrophages), the overall toxicity of the dissociated substance can be described by the toxicity of the individual constituents. Since synergistic effects are not expected, the human health hazard assessment of the assessment entity triiron bis(orthophosphate) consists of an individual assessment of the assessment entities iron cation and the phosphate anion. The iron cation and the phosphate anion are considered to represent the overall toxicity of triiron bis(orthophosphate) in a manner proportionate to the phosphate and the metal (represented by one of its readily soluble salts). Based on the above information, unrestricted read-across is considered feasible and justified.

The hazard information of the individual constituents was obtained from publicly available peer-reviewed risk assessment documents, such as EFSA opinions, WHO recommendations for human nutrition.

 

Iron

Animal data

In a repeated dose toxicity study with reproductive and developmental screening (according to OECD 422 and under GLP), iron(II)sulfate was administered to rats at doses of 30, 100, 300 and 1000 mg/kg bw/day via gavage (Pharmaceutical and Food Safety Bureau 2002).

General observation revealed salivation in males and females in the ≥300 mg/kg bw/day 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.

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 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.

 

Human data

There is extensive data available on the iron requirements of humans throughout their maturation to an adult.

The newborn term infant has an iron content of about 250–300mg (75mg/kg body weight). During the first 2 months of life, haemoglobin concentration falls because of the improved oxygen situation in the newborn infant compared with the intrauterine foetus. This leads to a considerable redistribution of iron from catabolized erythrocytes to iron stores.

In the term infant, iron requirements rise markedly after age 4–6 months and amount to about 0.7–0.9 mg/day during the remaining part of the first year. These requirements are very high, especially in relation to body size and energy intake (SCF, 1993).

The requirements for absorbed iron in infants and children are very high in relation to their energy requirements. For example, in infants 6–12 months of age, about 1.5 mg of iron need to be absorbed per 4.184 MJ and about half of this amount is required up to age 4 years. The prevention of iron deficiency has become more urgent in recent years with the accumulation of evidence strongly suggesting a relationship between even mild iron deficiency and impaired brain development, and especially so in view of the observation that functional defects affecting learning and behaviour cannot be reversed by giving iron at a later date.

Iron requirements are also very high in adolescents, particularly during the period of rapid growth (Rossander-Hulthén, 1996).

 

Iron requirements during pregnancy are well established. Most of the iron required during pregnancy is used to increase the haemoglobin mass of the mother; this increase occurs in all healthy pregnant women who have sufficiently large iron stores or who are adequately supplemented with iron. An adequate iron balance can be achieved if iron stores of 500 mg are available during the second and third trimesters. However, it is uncommon for women today to have iron stores of this size.

 

The physiological adjustments occurring in pregnancy are not sufficient to balance its very marked iron requirements, and the pregnant woman has to rely on her iron stores.

Based on the information presented above, there is no evidence of any developmental toxicity for iron compounds. Iron is abundantly available in the environment and in food, and is extensively distributed throughout the human body with a highly efficient regulatory capacity preventing iron overload in a state of increased dietary iron intake or bioavailability. Such characteristics of an essential trace element is contradictory to allegation of developmental toxicity potential.

Potential for developmental toxicity of the abundantly available essential element iron can be safely excluded. Therefore, further testing should not be considered, inter alia for reasons of animal welfare. In conclusion, conduct of studies for developmental toxicity on iron is considered to be scientifically unjustified (in accordance with regulation (EC) 1907/2006, Annex XI, Section 1.1.3 and 1.2).

 

Phosphate

A registration dossier shall contain information on the human health hazard assessment (regulation 1907/2006, Art.10). However, it is considered that the information requirements for orthophosphate as laid down in annex VII to IX can be fulfilled by adaptation of the standard testing regime according to Annex XI, points 1.1.3, 1.2, as presented in the following:

Phosphorus is most commonly found as the phosphate ion, with phosphorus in its pentavalent form. Thus, in the following the term phosphorus refers to phosphate, including its major form orthophosphate.

(1) A large part of human nutrition consists of phosphorus as cited by EFSA, 2015:

The major dietary contributors to phosphorus intake are foods high in protein content, i.e. milk and milk products followed by meat, poultry and fish, grain products and legumes. Based on data from 13 dietary surveys in nine European Union countries, mean phosphorus intakes range from 265 to 531 mg/day in infants, from 641 to 973 mg/day in children aged 1 to < 3 years, from 750 to 1202 mg/day in children aged 3 to < 10 years, from 990 to 1601 mg/day in children aged 10 to < 18 years and from 1000 to 1767 mg/day in adults (≥18 years).(EFSA, 2015)

 

(2) The EFSA and JECFA concluded on phosphorus:

The available data[derived from short and long term studies in rodents and humans, and summarised by EFSA, 2005]indicate that normal healthy individuals can tolerate phosphorus intakes up to at least 3000 mg phosphorus per day without adverse systemic effects. In some individuals, however, mild gastrointestinal symptoms, such as osmotic diarrhoea, nausea and vomiting, have been reported if exposed to supplemental intakes >750 mg phosphorus per day. Estimates of current intakes of phosphorus in European countries indicate total mean dietary and supplemental intakes around 1000-1500 mg phosphorus per day, with high (97.5 percentile) intakes up to around 2600 mg phosphorus per day. There is no evidence of adverse effects associated with the current intakes of phosphorus.(EFSA, 2005)

Long-term effects of dietary phosphoric acid in three generations of rats have been investigated (Bonting and Jansen, 1956). The animals received diets containing 1.4% and 0.75% phosphoric acid (equivalent to approximately 200 and 375 mg phosphorus/kg body weight/day) for 90 weeks. No harmful effects on growth or reproduction were observed, and also no significant differences were noted in haematological parameters in comparison with control animals. There was no acidosis, nor any change in calcium metabolism. The quality of these older studies would be considered limited by current standards. (EFSA, 2005)

JECFA reviewed the available data from studies in mice and rats and concluded that dosing with phosphoric acid and inorganic phosphate salts does not induce maternal toxicity or teratogenic effects. Maximum dose levels tested for the various inorganic phosphate salts varied between 130 and 410 mg phosphorus/kg bodyweight (JECFA, 1982).(EFSA, 2005)

(3) Exposure of breast-fed babies to phosphorus via mother milk: A breast-fed of 3 months age has an average weight of 6.5 kg (WHO 2013) and the infant ingests approx. 180mL/kg bw of milk per day (Riordan 2001), being 1170 mL for a baby at an age of 3 months. The phosphorus content of mother milk is approx. 140 mg/mL at 90 days post partum (Atkinson et al. 1995; EFSA, 2015). This results in a total “exposure” for a 3 month old baby of 163.8 mg phosphorus per day, being 25.2 mg/kg bw/day.

A detailed evaluation of the underlying single studies is not provided here, in order to avoid unnecessary duplication of the work already performed by an EU-nominated expert body. Based on the findings evaluated in the EFSA document, an upper intake level (UL) cannot be established based on the effect of a high phosphorus intake on the activity of calcium regulating hormones, which the expert body considers not to be adverse in themselves, and which have no demonstrable effects on bone mineral density and skeletal mass. (EFSA, 2005)

 

Based on the above given arguments, one may safely assume that human exposure towards phosphorus substances exerts any adverse effects of toxicological relevance after chronic exposure.

In conclusion, the conduct of any further toxicity studies with chronic exposure in animals would not contribute any new information and is therefore not considered to be required.

 

Triiron bis(orthophosphate)

Since no developmental toxicity study is available specifically for triiron bis(orthophosphate), information on the individual constituents iron and phosphate will be used for the hazard assessment and when applicable for the risk characterisation of triiron bis(orthophosphate). For the purpose of hazard assessment of triiron bis(orthophosphate), no hazard was identified for the individual assessment entities iron and phosphate. Consequently, no hazard is identified for the assessment entity triiron bis(orthophosphate) with regard to developmental toxicity.

 

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