Trimanganese bis(orthophosphate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Trimanganese bis(orthophosphate) does not show adverse effects towards male and female reproductive organs in oral repeated dose toxicity studies in animals.

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 trimanganese 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 manganese moiety (or one of its readily soluble salts). This way forward is acceptable, since trimanganese bis(orthophosphate) dissociates to the phosphate anion and the manganese cation upon dissolution in aqueous media.

Manganese exists in various oxidation states (i.e. -3 to +7), from which Mn²⁺and Mn³⁺were relevant in biological systems.As to the speciation of manganese under physiological conditions, Mn²⁺must be assumed to be the prevailing species under mildly acidic conditions according to the Pourbaix diagram for manganese.

In accordance with ATSDR, 2012:

Manganese is capable of existing in a number of oxidation states, and limited data suggest that inorganic manganese may undergo changes in oxidation state within the body. Circumstantial support for this hypothesis comes from the observation that the oxidation state of the manganese ion in several enzymes appears to be Mn(III) (Leach and Lilburn 1978; Utter 1976), while most manganese intake from the environment is either as Mn(II) or Mn(IV). Another line of evidence is based on measurements of manganese in tissues and fluids using electron spin resonance (ESR), which detects the unpaired electrons in Mn(II), Mn(III), and Mn(IV). When animals were injected with manganese chloride, levels of manganese increased in bile and tissues, but only a small portion of this was in a form that gave an ESR signal (Sakurai et al. 1985; Tichy and Cikrt 1972). This suggests that Mn(II) is converted to another oxidation state (probably Mn(III)), but it is also possible that formation of complexes between Mn(II) and biological molecules (bile salts, proteins, nucleotides, etc.) results in loss of the ESR signal without oxidation of the manganese ion. Evidence by Gibbons et al. (1976) suggests that oxidation of manganese occurs in the body. It was observed that human ceruloplasmin led to the oxidation of Mn(II) to Mn(III) in vitro, and although the process was not studied in vivo, it is a likely mechanism for manganese oxidation in the blood. These authors also noted that manganese oxidation led to a shift in manganese binding in vitro from α2-macroglobulin to transferrin and that in vivo clearance of Mn(II)-α2-macroglobulin from cows was much more rapid than the clearance of Mn(III)-transferrin (Gibbons et al. 1976). This suggests that the rate and extent of manganese reduction/oxidation reactions may be important determinants of manganese retention and toxicity in the body. Reaney et al. (2006) compared brain concentrations of manganese, dopamine, and gamma amino butyric acid in female retired breeder Long Evans rats exposed to cumulative intraperitoneal doses of 0, 30, or 90 mg manganese/kg of Mn(II) chloride or Mn(III) pyrophosphate. Rats were given intraperitoneal doses of 0, 2, or 6 mg manganese/kg, 3 times/week for 5 weeks. In Mn(III)-treated rats, brain manganese concentrations (analyzed in the striatum, globus pallidus, thalamus, and cerebrum regions) and blood concentrations were higher than brain concentrations in Mn(II)-treated rats. The only other marked changes in end points between the two treatment groups was that the highest Mn(III) exposure group showed a 60% increased dopamine level in the globus pallidus (compared with controls), whereas the comparably treated Mn(II) rats showed a 40% decrease in globus pallidus dopamine level. These results suggest that manganese valence state can influence tissue toxicokinetic behavior, and possibly toxicity.

 

 

Once the individual constituents of trimanganese 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 consists of an individual assessment of the manganese cation and the phosphate anion.

The hazard information of the individual constituents was obtained from publicly available literature (i.e. EFSA documents, ATSDR toxicological profile and WHO recommendations for human nutrition).

Trimanganese bis(orthophosphate) readily dissociates to the corresponding manganese cations and phosphate anions. The manganese cation and the phosphate anion are considered to represent the overall toxicity of trimanganese 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.

Manganese

In a chronic feeding study, male and female F344/N rats were given doses of 1500, 5000, and 15000 ppm of manganese (II) sulfate monohydrate (actual dose received: 60, 200, and 615 mg/kg/day for males and 70, 230, and 715 mg/kg/day for females, respectively). No adverse effects in male or female reproductive organs were reported. Based on the absence of any adverse effects related to the test substance, a No Observed Adverse Effect Level (NOAEL) for test item of 15000 ppm (actual dose received: 615 mg/kg/day for males and 715 mg/kg/day for females (200 and 233 mg Mn/kg/day, respectively) was concluded for male and female rats.

In a chronic feeding study, male and female B6C3F1 mice were given doses of 1500, 5000, and 15000 ppm of manganese (II) sulfate monohydrate (actual dose received: 160, 540, and 1800 mg/kg/day for males and 200, 700, and 2250 mg/kg/day for females, respectively). No adverse effects in male or female reproductive organs were reported. Based on the histopathological findings in the thyroid (follicular dilatation and focal hyperplasia), decreased final body weight in females, and haematological findings in the male mice at the 15000 ppm dose level, a No Observed Adverse Effect Level (NOAEL) for test item of 5000 ppm (actual dose received: 540 mg/kg/day for males and 700 mg/kg/day for females (172.8 and 224 mg Mn/kg/day, respectively)) was concluded for male and female mice.

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.

Trimanganese bis(orthophosphate)

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

References:

Agency for Toxic Substances and Disease Registry (ATSDR): Toxicological profile for manganese. U.S:

Departement of Health and Human Services, Public Health Service, ATSDR, 2012.

Gibbons RA, Dixon SN, Hallis K, et al. 1976. Manganese metabolism in cows and goats. Biochim Biophys Acta 444:1-10.

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.

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.

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.

Leach RM, Lilburn MS. 1978. Manganese metabolism and its function. World Rev Nutr Diet 32:123134.

Reaney SH, Bench G, Smith DR. 2006. Brain accumulation and toxicity of Mn(II) and Mn(III) exposures. Toxicol Sci 93(1):114-124.

Sakurai H, Nishida M, Yoshimura T, et al. 1985. Partition of divalent and total manganese in organs and subcellular organelles of MnCl2-treated rats studied by ESR and neutron activation analysis. Biochim Biophys Acta 841:208-214.

Tichy M, Cikrt M. 1972. Manganese transfer into the bile in rats. Arch Toxikol 29:51-58.

Utter MF. 1976. The biochemistry of manganese. Med Clin North Am 60:713-727.

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

Trimanganese bis(orthophosphate) does not show adverse effects towards the developing organism in oral repeated dose toxicity studies in animals.

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 trimanganese 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 manganese moiety (or one of its readily soluble salts). This way forward is acceptable, since trimanganese bis(orthophosphate) dissociates to the phosphate anion and the manganese cation upon dissolution in aqueous media.

Manganese exists in various oxidation states (i.e. -3 to +7), from which Mn²⁺and Mn³⁺were relevant in biological systems.As to the speciation of manganese under physiological conditions, Mn²⁺must be assumed to be the prevailing species under mildly acidic conditions according to the Pourbaix diagram for manganese.

In accordance with ATSDR, 2012:

Manganese is capable of existing in a number of oxidation states, and limited data suggest that inorganic manganese may undergo changes in oxidation state within the body. Circumstantial support for this hypothesis comes from the observation that the oxidation state of the manganese ion in several enzymes appears to be Mn(III) (Leach and Lilburn 1978; Utter 1976), while most manganese intake from the environment is either as Mn(II) or Mn(IV). Another line of evidence is based on measurements of manganese in tissues and fluids using electron spin resonance (ESR), which detects the unpaired electrons in Mn(II), Mn(III), and Mn(IV). When animals were injected with manganese chloride, levels of manganese increased in bile and tissues, but only a small portion of this was in a form that gave an ESR signal (Sakurai et al. 1985; Tichy and Cikrt 1972). This suggests that Mn(II) is converted to another oxidation state (probably Mn(III)), but it is also possible that formation of complexes between Mn(II) and biological molecules (bile salts, proteins, nucleotides, etc.) results in loss of the ESR signal without oxidation of the manganese ion. Evidence by Gibbons et al. (1976) suggests that oxidation of manganese occurs in the body. It was observed that human ceruloplasmin led to the oxidation of Mn(II) to Mn(III) in vitro, and although the process was not studied in vivo, it is a likely mechanism for manganese oxidation in the blood. These authors also noted that manganese oxidation led to a shift in manganese binding in vitro from α2-macroglobulin to transferrin and that in vivo clearance of Mn(II)-α2-macroglobulin from cows was much more rapid than the clearance of Mn(III)-transferrin (Gibbons et al. 1976). This suggests that the rate and extent of manganese reduction/oxidation reactions may be important determinants of manganese retention and toxicity in the body. Reaney et al. (2006) compared brain concentrations of manganese, dopamine, and gamma amino butyric acid in female retired breeder Long Evans rats exposed to cumulative intraperitoneal doses of 0, 30, or 90 mg manganese/kg of Mn(II) chloride or Mn(III) pyrophosphate. Rats were given intraperitoneal doses of 0, 2, or 6 mg manganese/kg, 3 times/week for 5 weeks. In Mn(III)-treated rats, brain manganese concentrations (analyzed in the striatum, globus pallidus, thalamus, and cerebrum regions) and blood concentrations were higher than brain concentrations in Mn(II)-treated rats. The only other marked changes in end points between the two treatment groups was that the highest Mn(III) exposure group showed a 60% increased dopamine level in the globus pallidus (compared with controls), whereas the comparably treated Mn(II) rats showed a 40% decrease in globus pallidus dopamine level. These results suggest that manganese valence state can influence tissue toxicokinetic behavior, and possibly toxicity.

 

 

Once the individual constituents of trimanganese 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 consists of an individual assessment of the manganese cation and the phosphate anion.

The hazard information of the individual constituents was obtained from publicly available literature (i.e. EFSA documents, ATSDR toxicological profile and WHO recommendations for human nutrition).

Trimanganese bis(orthophosphate) readily dissociates to the corresponding manganese cations and phosphate anions. The manganese cation and the phosphate anion are considered to represent the overall toxicity of trimanganese 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.

Manganese

In a supporting study female weanling Sprague-Dawley rats were randomly divided in six groups with 17 animals per group and fed manganese sulfate in the diet at 0, 4, 24, 54, 154, 504 and 1004 mg/kg dry diet (Järvinen & Ahlström, 1975). After 8 weeks they were mated with males of the same strain and after 21 days the animals and fetuses were killed and examined. No treatment related effects were seen on number and percent of live and dead foetuses, resorptions and pre- and post-implantation losses. No gross malformation, bone structure abnormality or altered fetal weight was observed. No information on the sex ratio given. An increase in fetal manganese concentration could only be seen in the highest dose (1004 mg/kg). There was no maternal toxicity.

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.

Trimanganese bis(orthophosphate)

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

References:

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.

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.

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.

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

Justification for classification or non-classification

There is no reliable equivocal animal evidence to link trimanganese bis(orthophosphate) with reproductive toxicity via relevant routes of exposure. The NTP (1993) report (see IUCLID section 7.5.1) showed no effect on the testes of rats exposed orally for up to 2 years. There is no reliable evidence to suggest that trimanganese bis(orthophosphate) is a developmental toxicant. Järvinen R & Ahlström A (1975) found no adverse developmental effects via the oral route in a rat study.