Triiron bis(orthophosphate)

Endpoint summary

Description of key information

Triiron bis(orthophosphate) does not show adverse effects in oral repeated dose toxicity studies in animals. Extensive information on an absence of severe adverse effects in humans is available. Solely adverse gastro-intestinal effects are observed after acute ingestion of extremely high doses of (soluble) non-haem iron substances.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

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

Considering the role of iron in human metabolic processes, it is highlighted that iron has several vital functions in the body. It serves as a carrier of oxygen to the tissues from the lungs by red blood cell haemoglobin, as a transport medium for electrons within cells, and as an integrated part of important enzyme systems in various tissues. It is an essential constituent of oxygen carriers, such as haemoglobin and myoglobin, and the iron contained within haem is essential for the redox reactions of numerous cytochromes. Insufficient intake results in the deficiency condition anaemia, adverse outcomes of pregnancy, impaired psychomotor development and cognitive performance and reduced immune function.

Any elemental iron in the diet is probably absorbed as non-haem iron following its dissolution in the acid stomach contents. The absorption of non-haem iron can be increased substantially by the presence of ligands, such as ascorbate, citrate and fumarate, as well as the presence of amino acids (e.g. cysteine) and oligopeptides resulting from meat digestion (Mulvihill et al., 1998). In contrast, very stable complexes, for example with phytates, phosphates and oxalates, impair non-haem iron absorption. Depending on the concentration of supportive or inhibitory ligands in the intestinal lumen the absorption of non-haem iron can vary by a factor of 10 in single-meal studies, but the effects are less pronounced in more long-term studies (Hallberg and Rossander, 1984; Rossander, 1987; Hunt and Roughead 2000). Iron is reversibly stored within the liver as ferritin and haemosiderin whereas it is transported between different compartments in the body by the protein transferrin. Iron excretion via the kidneys is very low, and body iron is highly conserved. Renal elimination is not controlled as part of iron homeostasis or the control of excess body stores. Normally, only about 0.1 mg is lost daily in urine. The sloughing of mucosal enterocytes results in elimination of absorbed iron before it reaches the systemic circulation and accounts for the loss of 0.6mg per day into the intestinal lumen. About 0.2-0.3 mg is lost daily from the skin. The total daily loss is equivalent to about 0.05 % of body iron content (Green et al., 1968).

 

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.

After the administration 1000 mg/kg bw/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. Body weights in the 1000 mg/kg bw/day group were significantly lower throughout the administration period in the males

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 bw/day 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 bw/day group, and high absolute and relative liver weights in females in the 1000 mg/kg bw/day group.

Microscopical investigation revealed that the stomach findings are characterised as 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.

After the administration 300 mg/kg/day of the test item, merely blood biochemistry tests revealed slightly elevated organic phosphorus levels in females in the 300 mg/kg group, which is not considered being adverse.

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

 

In an unbound study by Appel et al. 2001, groups of 40 male Sprague-Dawley rats each were fed iron(II) sulfate supplemented diet, resulting in doses of 2.84, 5.69, or 11.54 mg Fe/kg bw/day. Twenty rats of each group were sacrificed after 31 days of feeding and 20 rats of each group were sacrificed after 61 days of feeding. The following parameters were measured: clinical signs, body weights, food consumption, food conversion efficiency, haematology, clinical chemistry as well as gross pathology, organ weights and histopathology of selected organs (liver, spleen and all gross lesions). A NOAEL for male rats of >11.54 mg Fe/kg bw/day was derived. The NOAEL is based on a lack of test substance-related effects on clinical signs, body weight, organ weights, food consumption, haematology, clinical chemistry, gross pathology, and histopathology.

 

In a range finding study for carcinogenicity testing, groups of 10 male and 10 female Fischer 344 rats were administered ferric chloride hexahydrate (FeCl3 6H2O) via drinking water ad libitum for a exposure period of 13 weeks (Sato et al. 1985). The dose levels were 0%, 0.12 %, 0.25 %, 0.5 %, 1.0 % or 2.0 % (equivalent to 80, 154, 277, 550, and 1231 mg/kg bw/day for males and 88, 176, 314, 571, and 1034 mg/kg bw/day for females, respectively). The following parameters were examined: clinical signs, mortality, body weight, water consumption, haematology, clinical chemistry, gross pathology, organ weights and histopathology. No mortality was observed during the course of the study. A significant suppression of 17 % to 60 % in the intake of drinking water was observed in the groups given doses of 0.5 % and above. Males of the treatment groups demonstrated a dose-dependent increase of serum iron levels. The histopathological examination revealed brown pigment deposition in the keratin layers of the oesophageal mucosa in the groups given doses of 0.25 % and above and in the laminae propriae of the large intestine in the 2.0 % group. A NOAEL of 277 mg/kg bw/day for the male and 314 mg/kg bw/day for the female animals was derived, based on reduction in the rate of body weight gain.

 

Human data

The side effects of oral iron preparations increase with increase in dosage, but there are fewer side effects with slow delivery systems or if the iron is taken with food (Brock et al., 1985; Reddaiah et al., 1989). The adverse gastrointestinal effects are related to the concentration of iron in the intestinal lumen (Cook et al., 1990).

A daily dose of 50 mg of iron produced a higher incidence of gastrointestinal effects in subjects given conventional ferrous sulphate compared with subjects given the same amount in a wax-matrix (Brock et al., 1985), and also in subjects given ferrous sulphate compared with subjects given the same amount of iron as bis-glycino iron (Coplin et al., 1991). Neither of these studies included a placebo group, and therefore the association with iron is based on different responses to different preparations. A higher incidence of side effects was reported in subjects given 60 mg of iron as iron fumarate daily compared with placebo; daily doses of 120 mg of iron as fumarate for 8 weeks given to 19 young women in a double blind cross-over study resulted in gastrointestinal effects in 5 subjects while receiving iron, compared with 2 while taking placebo (Frykman et al., 1994).

Iron overload with clinical symptoms, which has only been found in adult subjects homozygous for hereditary haemochromatosis, those under long-term, high-dose medical treatment with iron, and those given repeated blood transfusions. Such information is of no relevant for the chemicals safety assessment.

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 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)

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 repeated dose 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).

References

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