Reaction products of sodium glucoheptonate with iron sulfate and ammonium hydroxide

Endpoint summary

Description of key information

Acute toxicity:

To address the endpoint acute toxicity, read-across on gluconates and derivatives and iron compounds was performed within the frame of a weight-of-evidence approach. The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

The available data provide evidence that iron glucoheptonate is not acutely toxic via oral route. This conclusion is based on good quality data with different animal species on the main constituent iron glucoheptonate and the read-across sources iron gluconate and gluconates and derivatives. All these studies reveal LD50 values significantly > 2000 mg/kg bw when converted to the target substance. Regarding the inhalation route, the available studies indicate certain inhalatory toxicity of iron, when applied as nanoparticle. When evaluating these results, it has to be considered that the toxicity of nanoparticles arise rather from their increased reactivity due to their high surface to mass ratio than from their chemical constitution. In addition, iron glucoheptonate has no particles < 15 µm, which are able to reach the alveolar region. Furthermore, the toxic relevant iron ion is complexed by a sugar residue with a high molecular weight in the target substance iron glucoheptonate. Therefore, an enormous amount of the substance would have to be inhaled to reach an iron concentration in the lungs being toxically relevant. Therefore no hazard via inhalation is expected for iron glucoheptonate.

Based on the results of the available studies, the registered substance does not meet criteria for classification and labelling as a skin or eye irritant in accordance with European Regulation (EC) No 1272/2008.

Acute toxicity oral:

Data on iron glucoheptonate

The Commission Regulation No 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin lists in its Annex pharmacologically active substances and their classification regarding maximum residue limits (MRL), i.e. iron glucoheptonate. Iron glucoheptonate is marked: No MRL required.

Data on iron gluconate

Berenbaum et al. (1960) compared ferrous fumarate, ferrous sulphate A.R., ferrous succinate and ferrous gluconate B.P.C in their toxicologic and hematinic properties (Berenbaum, 1960). The ferrous iron contents of ferrous gluconate was 11.7 per cent. It was administered as aqueous suspensions containing 0.1 per cent w/v of tragacanth. In these acute oral toxicity tests in mice, groups of 10 male fawn mice (GFF strain, bodyweights 17 to 22 gr) were dosed orally and then observed for seven days, when the percentage mortalities were recorded. The LD₅₀values, which were calculated according to de Beer (1945) and expressed in mg Fe/Kg, were for fumarate LD₅₀= 630 mg Fe/kg, for succinate LD₅₀= 560 mg Fe/kg, for gluconate LD₅₀= 320 mg Fe/kg and for sulphate LD₅₀= 230 mg Fe/kg. Thus the relative toxicities were fumarate 1 , succinate 1 .1 , gluconate 2.0 and sulphate 2.7.

The LD50 value for ferrous gluconate itself was calculated taking into account the ferrous iron content given for this compound (11.7 %): LD₅₀= 2735 mg/kg bw.

Moreover, Berenbaum et al. (1960) investigated the irritant effects of orally administered very high amounts of ferrous gluconate on the gastric mucosa in rabbits (Berenbaum, 1960). Sixty-four adult rabbits having free access to food and water were dosed orally with tablets of ferrous fumarate, ferrous sulphate compound, ferrous succinate or ferrous gluconate. The dose was 450 mg Fe/Kg, the rabbits receiving seven tablets of fumarate or sulphate compound, or twelve tablets of succinate or gluconate per Kg. The fumarate and sulphate tablets contained 65 mg Fe, the succinate and gluconate tablets 36 mg.

In case of ferrous gluconate 3 of 9 animals died within 12 hours. The histologic findings in case of ferrous gluconate were as follows: the stomach of one revealed iron incrustation of the mucosa, while that of another showed superficial mucosal necrosis and iron in the vessels. In both, the pylorus showed early inflammation. Iron impregnation was apparent in the lamina propria of one, and there was iron in the vessels of the other. The livers showed a “chemical hepatitis,” the essential features of which were iron impregnation of parenchymal cells, their invasion and replacement by polymorphs, and an increase in intravascular polymorphs.

In a second experiment, four groups of three adult rabbits were dosed orally with tablets of the four iron compounds. As before, the dose employed was 450 mg Fe/Kg. One of the three animals died within 12 hours of being dosed.

The report of Weaver et al. (1961) is concerned with the animal toxicities of a new high molecular iron-carbohydrate complex compared to those of several other preparations, i.e. ferrous gluconate (Weaver, 1961). For this purpose several toxicity tests were conducted in different animal species. In the acute toxicity test in mice and rats the compounds (i.e. ferrous gluconate) were administered as aqueous solutions where possible, otherwise as fine suspensions.

Groups of 10 or more male albino Swiss-Webster mice were given the compounds intravenously (i.v.), intraperitoneally (i.p.), or intragastrically (i.g.). The rate of i.v. injections was 0.01 mL per second. The animals were observed closely for several hours following injection, and the LD₅₀ and 95% confidence limits were determined at the end of 24 hours by the method of Litchfield and Wilcoxon. The animals receiving iron-carbohydrate complex and ferrous sulfate were observed for a period of 7 days following injection and any delayed manifestations of toxicity were recorded. If any deaths occurred after 24 hours, the LD₅₀ was recalculated at the end of the 7-day observation period. Strain and sex differences in response to iron-carbohydrate complex and ferrous sulfate were evaluated by the use of female albino Swiss-Webster, black female BDF-1 and C-57 and female fawn DBA-2 mice.

Morover, male (250 to 350 g) and female (150 to 250 g) Harlan-Wistar rats in mixed groups of 6 received the iron compounds i.g. in attempts to determine 24-hour LD50 in this species. The animals were observed closely for several hours following injection, and the LD₅₀ and 95% confidence limits were determined at the end of 24 hours by the method of Litchfield and Wilcoxon.

Since the iron content of the compounds varies considerably comparisons were made on the basis of actual iron content.

Intragastrically, iron-carbohydrate complex was at least 6 times less toxic than any of the other compounds tested. The volumes necessary made it impractical to attempt to determine the i.g. toxicity of the iron polysaccharide complex. There was no significant difference between the 1- and 7-day toxicities for either iron-carbohydrate complex or ferrous sulfate for the i.v. and i. g. routes in mice or rats. In Swiss-Webster male mice the i.g. LD₅₀ was 3950 mg/kg for ferrous gluconate. The i.g. LD₅₀ of iron-carbohydrate complex in male Swiss-Webster was >8000 mg/kg. There is no evidence of sex or strain differences in these very limited studies.

In rats, none of the compounds was very toxic following i.g. administration and evidently there are only slight differences in the lethal effects of ferrous sulfate, ferrous gluconate (LD50 = 7460 mg/kg) and ferroglycine sulfate complex.

Thus, iron-carbohydrate complex showed a low order of toxicity in 3 species of laboratory animals. Studies in mice indicate that iron-carbohydrate complex is less toxic than ferrous sulfate, ferrous gluconate, ferrous fumarate, ferroglycine sulfate complex, ferric choline citrate and iron polysaccharide complex by the oral and intraperitoneal routes. In addition, none of the compounds tested was less toxic than iron-carbohydrate complex by the oral route in rats.

 

Hoppe et al. (1955) determined firstly the acute toxicity of ferrous gluconate in direct comparison with that of ferrous sulfate following both intravenous and oral administration in male albino Swiss mice weighing 22 ± 2 g and in male Sprague-Dawley rats weighing 100 ± 10 g (Hoppe, 1955). A volume of 0.01 cc/g of body weight in aqueous solution was used for oral administration. All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The mice and rats were fasted for 4 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The animals were observed closely for several hours following injection, and the LD₅₀ ± SE was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD50 was recalculated at the end of the 7-day observation period.

The acute oral toxicity data show that ferrous gluconate is significantly less toxic than ferrous sulfate, both in terms of the salt and of ferrous iron. The oral LD₅₀ for ferrous sulfate was found to be 1520 ± 130 mg/kg compared with a LD₅₀ of 3700 ± 145 mg/kg of ferrous gluconate which, when expressed in terms of ferrous iron, amounts to 306 mg/kg as the sulfate and 429 mg Fe /kg as the gluconate. These differences are statistically significant and indicate that the gluconate, in terms of ferrous iron content, is approximately 40% better tolerated than the sulfate. There were no delayed deaths with either compound following oral administration in mice.

No delayed deaths of significance were observed following oral administration in either species.

The acute oral toxicity data in the rat were found to be of a similar order of magnitude as those found in the mouse. Studies indicate that the acute oral toxicity of ferrous gluconate (LD₅₀ of 4600 ± 560 after 24 hours or 4500 ± 400 mg/kg after 7 days) was only one-third as toxic as ferrous sulfate (1480 ± 184 mg/kg after 24 hours or 7 days) as the salt and one-half as toxic in terms of ferrous iron. No delayed deaths were observed following oral administration of ferrous sulfate; one delayed death was observed with ferrous gluconate.

 

Moreover, Hoppe et al. (1955) investigated the acute toxicity of ferrous gluconate in direct comparison with that of ferrous sulfate following oral administration in cats and mongrel dogs (Hoppe, 1955). Ferrous gluconate and ferrous sulfate were administered orally as a finely divided powder by capsule to cats, weighing 2 to 3 kg, and to mongrel dogs weighing 7 to 12 kg in an effort to determine the acute lethal dose following oral administration.

Five dogs for ferrous sulfate and six dogs for ferrous gluconate, one at each dosage level (50, 100, 200, 400 and 800 mg/kg for ferrous sulfate and 100, 200, 400, 800, 1600 and 3200 mg/kg for ferrous gluconate), were given capsules of finely divided ferrous gluconate in amounts ranging from 100 to 3200 mg/kg. Cats received doses of 200 mg/kg ferrous sulfate and 400 mg/kg of ferrous gluconate. All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The cats and dogs were fasted for 18 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The cats and dogs were observed closely for several hours following injection, and the LD₅₀ ± SE was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD₅₀ was recalculated at the end of the 7-day observation period.

In cats, it was not possible to obtain mortality data by this route of administration at the dose levels employed, since emesis occurred in every cat within 15 minutes to one hour after medication. Severe diarrhea was also observed, but became less evident at the higher dosages as the promptness and intensity of emesis increased. It was concluded from these experiments that the acute oral lethal dose of ferrous sulfate in cats was more than 200 mg/kg (LD₅₀ > 200 mg/kg) and more than 400 mg/kg for ferrous gluconate (LD₅₀ > 400 mg/kg). The pattern of emesis was sufficiently prominent and consistent to permit the estimation of the approximate median emetic dose, AED₅₀, (the approximate dose producing emesis in 50% of the cats) as a criterion for comparing the gastric tolerance to these two compounds in cats. It will be noted that the dose of ferrous gluconate required to produce emesis in 50% of the cats was more than three times as large as that of ferrous sulfate. About twice as much iron in the form of ferrous gluconate was tolerated without vomiting as was tolerated in the form of the sulfate.

In dogs, no deaths or serious evidence of acute systemic intoxication were observed at doses up to and including the highest dose level, 800 mg/kg of ferrous sulfate or 3200 mg/kg of ferrous gluconate. The most obvious effects produced by these two compounds were emesis and diarrhea. Vomiting was noted in the dog receiving 50 mg/kg of ferrous sulfate but was not encountered in the others until the dose was raised to 800 mg/kg, when a prompt and vigorous emetic reaction was observed. With ferrous gluconate, vomiting did not occur until doses of 1600 and 3200 mg/ kg were reached. A watery diarrhea became apparent approximately one hour after oral administration of 100 mg/kg of ferrous sulfate and 800 mg/ kg of ferrous gluconate. At doses of 200 and 400 mg/kg of ferrous gluconate, diarrhea developed the morning of the day following medication. The occurrence of vomiting and diarrhea, indicative of a protective mechanism similar to that observed in the cat, interfered with the attempt to estimate the acute oral lethal dosage of these compounds in dogs.

In summary, the acute oral median lethal dose in the dog was estimated to be greater than 800 mg/kg of ferrous sulfate and more than 3200 mg/kg of ferrous gluconate (LD50 > 3200 mg/kg). No deaths or serious evidence of acute systemic intoxication were observed at these doses. The emesis and diarrhea produced by both compounds rendered attempts to estimate accurate LD₅₀ values impracticable.

The magnitude of the acute oral toxicity values when compared with the acute intravenous figures in mice indicates a relatively low order of absorption from the intestinal tract. An additional safety factor is evident from the oral studies in the cat and the dog in which the local irritant effects induce a protective emesis. These data suggest prompt, gentle gastric lavage along with supportive therapy for shock as an effective emergency measure in those cases where, for any reason, vomiting does not occur spontaneously following oral ingestion of ferrous sulfate, ferrous gluconate or other soluble iron salts.

In addition, an assessment report (Antula Healthcare AB) concerns Fexin effervescent tablets which contains Iron (II) as ferrous gluconate (each containing 80.5 mg Iron (II)) and which were approved through DCP (DK/H/1074/001/DC) on 3 October 2007 with Denmark as RMS. Based on the review of the data on quality, safety and efficacy, it was considered that the application for Fexin indicated for: iron deficiency anaemia, iron deficiency and for prophylactic use in pregnancy and for blood donors could be approved.

in this report a LD₅₀ value of 2200 mg/kg for dogs and a LD₅₀ value of 4600 mg/kg for rats is mentionned, however, no further reference is given.

The World Health Organisation evaluated ferrous gluconate already in 1975 (WHO, Technical Report No 576,1975). This compound was evaluated by the Committee not in relation to its use as a nutritional supplement but as a colouring adjunct. It was given an " ADI not specified ", with the proviso that "the contribution from ferrous gluconate to the total dietary gluconic acid intake from all sources should be included in the ADI for gluconic acid". Specifications were prepared at the eighteenth meeting of the Committee.

All of these results are also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Data on gluconates and derivatives

Data on acute oral toxicity for sodium gluconate in rat (Mochizuki, M, Bozo Research Center 1995) (doses: 500, 1000, 2000 mg/kg) and dog (Okamoto M., 1995) (doses: 1000 and 2000 mg/kg) fed by gavage showed no death at any dose, hence the minimum lethal dose was estimated > 2000 mg/kg for both species.

Rats were fed by gavage 3000, 3600, 4320, 5190, 6210 mg/kg bw (30% (w/v) aqueous solution) potassium gluconate and were observed for signs of toxicity during a 14-day period. One animal died in the 5190 mg/kg bw group and four animals in the 6210 mg/kg bw group. Deaths occurred between 5 and 21 hours after treatment. Survivors recovered gradually. The LD₅₀ was calculated (according to the method of Weil) to be 6060 mg/kg bw. However, the effects that were observed occurred at doses that exceed the accepted limit dose of 5000 mg/kg bw and the LD₅₀ may be related to high dosing (TNO, 1978).

Conclusion Studies with sodium gluconate in the rat and dog report LD50 values > 2000 mg/kg bw for both species. A gavage study with potassium gluconate and rats reported an LD50 of 6060 mg/kg bw.

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not toxic after oral intake is important.

Finally, the Wolrd Health Organisation evaluated gluconic acid and its derivatives (WHO, 1996, 1999). Consideration of glucono-delta-lactone and gluconic acid is based mainly on the metabolic evidence that these compounds are intermediates in a normal pathway of glucose metabolism in mammalian species. There is also considerable experience with the comparatively low toxicity of gluconate to man and animals.

Glucono-delta-lactone and gluconic acid are not toxic to animals and humans when given at very high dose levels (> 2000 mg/kg bw).

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that glucono-delta-lactone and gluconic acid are not toxic after oral intake is important.

Acute toxicity inhalation:

The particle size distribution of iron glucoheptonate was measured with sieves (USP method; Dabeer, 2011). The majority of the particles (87.5 %) has a size between 100 and 800 µm. 12.1 % of the particles are smaller than 100 µm but bigger than 40 µm. 0.1 % of the particles are smaller than 40 µm. Thus, aspiration of particles of iron glucoheptonate cannot be fully ruled out. A small amount of the particles may be able to reach the thoraic region, but there are no particles smaller than 15 µm. Therefore, iron glucoheptonate does not partition into the alveolar region even if inhaled.

Data on ferric oxide and iron oxide fumes

The toxic effects of inhalation exposure to ferric oxide (Fe₂O₃) nanoparticles in rats were investigated (Wang el al, 2010). Male Wistar rats were consecutively treated with Fe₂O₃ at 8.5 mg/kg body weight, twice daily tor 3 days. Fe₂O₃ nanoparticles were sprayed directly into both nasal passages using a dry powder sprayer. Content of Fe₂O₃ in tissues, biochemical parameters in serum, and histopathological examinations were analyzed at 12 h and 36 h after the 3 day treatment. An extended set of biochemical parameters was measured in serum, which was obtained from an abdominal vein at sacrifice. Blood serum was collected for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), albumin (ALB), and globulin (GLB) by an automatic biochemical analyzer (7170A, Hitachi, Tokyo). The content of Fe in lung, liver, kidney, olfactory bulb, and brain tissues were determined by neutron activation analysis (NAA). Changes in tissue pathology were also investigated to obtain information on the range of pathological changes that might occur following acute exposure to Fe₂O₃ nanoparticles.

After administration of Fe₂O₃ nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed. However, no deaths occurred over the entire duration of the experiment. In the Fe₂O₃-treated group, iron (Fe) content in liver and lung tissues was significantly increased at 36 h. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), and lactate dehydrogenase (LDH) in both nanoparticle-exposed groups were significantly decreased compared to the unexposed controls. Histopathological examination showed that the nanoparticles caused severe damage in liver and lung tissues. Inflammation, interstitial hyperemia, fatty degeneration around central vein, and hepatocyte necrosis were noticeable in the liver. Inflammation, interstitial hyperemia, emphysema, and interstitial substance hyperplasia were evident in the lung. There were no abnormal pathological changes in the trachea, kidney, or brain. Although this damage progressed in both liver and lung throughout the postexposure period, no significant elevation of serum enzyme activities was observed in response to either nanoparticle type.

These results show that once Fe₂O₃ nanoparticles entered the body via inhalation, it became systemically available and caused toxic effects in internal organs other than in the lungs. Pulmonary retention, extrapulmonary translocation, and redistribution were considered to be the essential mechanisms of organ damage induced by inhaled nanoparticles. Obvious lesions of liver and lung were induced and the levels of serum ALT, AST, ALP, CK, and LDH were all significantly decreased compared with the control groups.

Additionally, Sotiruou et al. (2012) investigated ferric oxide nanoparticles using a novel method which is suitable for assessing in-vivo the link between the physicochemical properties of engineered nanomaterials (ENMs) and their biological outcomes. The primary particle size for Fe₂O₃ was controlled from 4 to 25 nm, while the corresponding agglomerate mobility diameter of the aerosol was also controlled and varied from 40 to 120 nm. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENMs in intact animal models was also demonstrated using in-vivo chemiluminescence (IVCL). The IVCL technique is a highly sensitive method for identifying cardiopulmonary responses to inhaled ENMs under relatively small doses and acute exposures.

It was shown that moderate and acute exposures to inhaled nanostructured Fe₂O₃ can cause both pulmonary and cardiovascular effects.

In the studies of Wang et al. (2010) and Sotiriou et al. (2012) iron oxide nanoparticles were used as test substances. An extrapolation from the toxicity of the iron nanoparticles to the toxic effect of the chelated iron ions of iron glucoheptonate is difficult, because of the special toxicological behaviour of nanoparticles. Due to the high surface to mass ratio, nanoparticles are more reactive than larger-sized particles of the same chemistry (Oberdörster et al., 2005). In addition, other absorption routes are accessible for nanoparticles. "When inhaled, they are efficiently deposited in all regions of the respiratory tract; they evade specific defense mechanisms; and they can translocate out of the respiratory tract via different pathways and mechanisms (endocytosis and transcytosis)" (Oberdörster et al., 2005).

The majority of the iron glucoheptonate particles (87.5 %) has a size between 100 and 800 µm. 12.1 % of the particles are smaller than 100 µm but bigger than 40 µm. 0.1 % of the particles are smaller than 40 µm. Nanoparticles, in contrast have a size between 1 and 100 nm. So, the toxicological profile of iron glucoheptonate will differ significantly from the one of Fe2O3 nanoparticles. The inhalatary absorption rate is expected to be markedly reduced when compared to Fe2O3 nanoparticles. In addition, the high reactivity up to the increased surface to mass ratio is not relevant for iron glucoheptonate. Thus, iron oxide nanoparticles are expected to be much more toxic than iron glucoheptonate.

Furthermore, in the target substance iron glucoheptonate the iron is complexed by a sugar residue with a high molecular weight.

The National Institute for Occupational Safety and Health evaluated iron oxide fumes in 1978 (NIOSH, 1978). The Permissible Exposure Limit (PEL) for iron oxide fume is 10 mg/m3 air (8 h exposure) (OSHA standard). The Threshold Limit Value of iron oxide fume is 5 mg/m3 air (Recommendation of the American Conference of Governmental Industrial Hygienists).

Acute toxicity dermal:

There is no acute hazard for Fe glucoheptonate expected via dermal route of exposure. No hazard can be attributed to glucoheptonate moiety. Glucoheptonate is a sugar anologue and was assessed as safe for veterinary and medicinal products (EMEA, 1998). Glucoheptonate is used as imaging agent in patients with brain, renal and pulmonal tumors (Waxman et al. 1975, Boyd et al. 1973, Passamonte et al. 1983). No acute dermal hazard can be attributed to Fe cation (SIDS 2007). Moreover, absorption of Fe glucoheptonate in its chelated form and as Fe cation and glucoheptonate anion is neglectable via the skin. No significant amounts of Fe or glucoheptonate can be absorbed into systemic circulation (please refer to the toxicokinetic endpoint). Based on these considerations, dermal acute toxicity study is not necessary.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

other: Commision Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Commision Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Commission Regulation No 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. In its Annex there are pharmacologically active substances and their classification regarding maximum residue limits (MRL) listed, i.e. iron glucoheptonate.

GLP compliance:

no

Species:

other: not applicable - MRL value given in regulation valid all food producing species

Route of administration:

oral: unspecified

Sex:

male/female

Dose descriptor:

other: Maximum residue limit

Remarks on result:

other: No MRLrequired

Executive summary:

The Commission Regulation No 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. In its Annex there are pharmacologically active substances and their classification regarding maximum residue limits (MRL) listed, i.e. iron glucoheptonate,for which is marked: No MRL required.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Dose descriptor:

LD50

Effect level:

320 mg/kg bw

Based on:

element

Sex:

male

Dose descriptor:

LD50

Effect level:

2 735 mg/kg bw

Based on:

test mat.

Remarks:

Ferrous gluconate

Remarks on result:

other: calculated according to the ferrous iron content (11.7%) given for ferrous gluconate

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The LD50 value for ferrous gluconate was calculated taking into account the ferrous iron content given for this compound (11.7 %) and is 2735 mg/kg bw.
This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

The substances compared in these acute oral toxicity tests in mice were ferrous fumarate, ferrous sulphate A.R., ferrous succinate and ferrous gluconate B.P.C (Berenbaum, 1960). The ferrous iron contents of ferrous gluconate was 11.7 per cent. It was administered as aqueous suspensions containing 0.1 per cent w/v of tragacanth.

Groups of 10 male fawn mice (GFF strain, bodyweights 17 to 22 gr) were dosed orally and then observed for seven days, when the percentage mortalities were recorded. The LD50 values, which were calculated according to do Beer (1945) and expressed in mg Fe/Kg, were fumarate 630, succinate 560, gluconate 320 and sulphate 230. Thus the relative toxicities were fumarate 1 , succinate 1 . 1 , gluconate 2.0 and sulphate 2.7.

The LD50 value for ferrous gluconate was calculated taking into account the ferrous iron content given for this compound (11.7 %) and is 2735 mg/kg bw.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

not specified

Dose descriptor:

other: Concentration inducing the effects specified under 'Remarks'

Effect level:

450 mg/kg bw

Based on:

element

Remarks on result:

other:

Remarks:

The stomach of one revealed iron incrustation of the mucosa, while that of another showed superficial mucosal necrosis and iron in the vessels. In both, the pylorus showed early inflammation. Iron impregnation was apparent in the lamina propria of one, and there was iron in the vessels of the other. The livers showed a “chemical hepatitis,” the essential features of which were iron impregnation of parenchymal cells, their invasion and replacement by polymorphs, and an increase in intravascular polymorphs.

Sex:

not specified

Dose descriptor:

other: Concentration inducing the effects specified under 'Remarks'

Effect level:

3 232.09 mg/kg bw

Based on:

test mat.

Remarks:

converted to iron glucoheptonate

Remarks on result:

other:

Remarks:

The stomach of one revealed iron incrustation of the mucosa, while that of another showed superficial mucosal necrosis and iron in the vessels. In both, the pylorus showed early inflammation. Iron impregnation was apparent in the lamina propria of one, and there was iron in the vessels of the other. The livers showed a “chemical hepatitis,” the essential features of which were iron impregnation of parenchymal cells, their invasion and replacement by polymorphs, and an increase in intravascular polymorphs.

Mortality:

3/9

Clinical signs:

4/9 animals showed inflamation of the gastric mucosa (12 hours after giving a single dose equivalent to 450 mg Fe/kg). One of them slight and 3 of them severe and extensive inflammation

Body weight:

not examined

Gross pathology:

Gluconate: The stomach of one revealed iron incrustation of the mucosa, while that of another showed superficial mucosal necrosis and iron in the vessels. In both, the pylorus showed early inflammation. Iron impregnation was apparent in the lamina propria of one, and there was iron in the vessels of the other. The livers showed a “chemical hepatitis,” the essential features of which were iron impregnation of parenchymal cells, their invasion and replacement by polymorphs, and an increase in intravascular polymorphs.

The macroscopic changes in the gastric mucosa were classified according to the scheme in table 1, which includes the mortality figures.

The histologic findings were as follows:

Gluconate.-The stomach of one revealed iron incrustation of the mucosa, while that of another showed superficial mucosal necrosis and iron in the vessels. In both, the pylorus

showed early inflammation. Iron impregnation was apparent in the lamina propria of one, and there was iron in the vessels of the other. The livers showed a “chemical hepatitis,” the essential features of which were iron impregnation of parenchymal cells, their invasion and replacement by polymorphs, and an increase in intravascular polymorphs.

In a second experiment, four groups of three adult rabbits were dosed orally with tablets of the four iron compounds. As before, the dose employed was 450 mg Fe/Kg.

On this occasion none of the animals died within 12 hours of being dosed. They were all killed at this stage, and their stomachs were removed, washed with saline, and photographed.

Table 1 -Effects of 4 Different Iron Tablets on Rabbit Gastric Mucosa (12 hours after giving a single dose equivalent to 450 mg Fe/kg)
Effects observed	Ferrous gluconate
None	2/9
Slight inflammation	1/9
Severe and extensive inflammation	3/9
Death within 12 hours	3/9

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

The animal experiments were designed to compare the toxicologic and hematinic properties of ferrous fumarate with those of ferrous sulphate A.R.,ferrous succinate and ferrous gluconate B.P.C (Berenbaum, 1960). The ferrous iron contents of these four salts are 33.0, 20.0, 24.8 and 11.7 per cent, respectively. Sixty-four adult rabbits having free access to food and water were dosed orally with tablets of ferrous fumarate, ferrous sulphate compound, ferrous succinate or ferrous gluconate. The dose was 450 mg Fe/Kg, the rabbits receiving seven tablets of fumarate or sulphate compound, or twelve tablets of succinate or gluconate per Kg. The fumarate and sulphate tablets contained 65 mg Fe, the succinate and gluconate tablets 36 mg. The gluconate tablets were broken to facilitate administration; the other tablets were administered whole.

The tablets were administered at 10 p.m. and the mortalities were recorded at 10 a.m. next morning. The surviving rabbits were then killed, and the stomachs and livers from all the animals were examined macroscopically and those from three of each group histologically. The macroscopic changes in the gastric mucosa were classified according to the scheme in Table 1, which includes the mortality figures.

The macroscopic changes in the gastric mucosa were classified according to the scheme in table 1, which includes the mortality figures.

The histologic findings were as follows:

Gluconate.-The stomach of one revealed iron incrustation of the mucosa, while that of another showed superficial mucosal necrosis and iron in the vessels. In both, the pylorus

showed early inflammation. Iron impregnation was apparent in the lamina propria of one, and there was iron in the vessels of the other. The livers showed a “chemical hepatitis,” the essential features of which were iron impregnation of parenchymal cells, their invasion and replacement by polymorphs, and an increase in intravascular polymorphs.

In a second experiment, four groups of three adult rabbits were dosed orally with tablets of the four iron compounds. As before, the dose employed was 450 mg Fe/Kg. This corresponds to a concentration of 3232.09 mg/kg bw of iron glucoheptonate.

On this occasion none of the animals died within 12 hours of being dosed. They were all killed at this stage, and their stomachs were removed, washed with saline, and photographed.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

male

Dose descriptor:

LD50

Effect level:

3 950

Based on:

test mat.

95% CL:

>= 3 543 - <= 4 404

Remarks on result:

other: 3950 (3543-4404) mg/kg (= 457.4 Fe), as the Fe content of ferrous gluconate is 11.58 %

Results

The results in mice are summarized in Table 1.

Table 1 - Acute Toxicities Of Iron Compounds In Mice
LD50 (19 -20 Confidence Limits), mg/kg
		i.v. route			i.p. route			i.g. route
Compound	%Fe	No. mice	As salt	As Fe	No. mice	As salt	As Fe	No. mice	As salt	As Fe
Ferrous gluconate	11.58	55	199 (182-218)	23	30	160 (94-272)	18.5	100	3950 (3543-4404)	457.4

Since the iron content of the compounds varies considerably comparisons were made on the basis of actual iron content.

Intragastrically, iron-carbohydrate complex was at least 6 times less toxic than any of the other compounds tested. The volumes necessary made it impractical to attempt to determine the i.g. toxicity of the iron polysaccharide complex.

Summary.

Studies in mice indicate that iron-carbohydrate complex is less toxic than ferrous sulfate, ferrous gluconate, ferrous fumarate, ferroglycine sulfate complex, ferric choline citrate and iron polysaccharide complex by the oral and intraperitoneal routes. The iron polysaccharide complex by the intravenous route was the least toxic and iron-carbohydrate complex was next least toxic.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The LD50 determined (3950 mg/kg) showed the test item not to be toxic after single oral administration.
This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

This report is concerned with the animal toxicities of a new high molecular iron-carbohydrate complex compared to those of several other preparations (Weaver, 1961). For this purpose several toxicity test were conducted in different animal species. In the acute toxicity test in mice the compounds (i.e. ferrous gluconate) were administered as aqueous solutions where possible, otherwise as fine suspensions. Groups of 10 or more male albino Swiss-Webster mice were given the compounds intravenously (i.v.), intraperitoneally (i.p.), or intragastrically (i.g.). The rate of i.v. injections was 0.01 mL per second. The animals were observed closely for several hours following injection, and the LD50 and 95% confidence limits were determined at the end of 24 hours by the method of Litchfield and Wilcoxon. The animals receiving iron-carbohydrate complex and ferrous sulfate were observed for a period of 7 days following injection and any delayed manifestations of toxicity were recorded. If any deaths occurred after 24 hours, the LD50 was recalculated at the end of the 7-day observation period. Strain and sex differences in response to iron-carbohydrate complex and ferrous sulfate were evaluated by the use of female albino Swiss-Webster, black female BDF-1 and C-57 and female fawn DBA-2 mice.

Since the iron content of the compounds varies considerably comparisons were made on the basis of actual iron content. Intragastrically, iron-carbohydrate complex was at least 6 times less toxic than any of the other compounds tested. The volumes necessary made it impractical to attempt to determine the i.g. toxicity of the iron polysaccharide complex. There was no significant difference between the 1- and 7-day toxicities for either iron-carbohydrate complex or ferrous sulfate for the i.v. and i. g. routes in mice. In Swiss-Webster male mice the i.g. LD50 was 3950 mg/kg for ferrous gluconate. The i.g. LD50 of iron-carbohydrate complex in male Swiss-Webster was >8000 mg. per kg. There is no evidence of sex or strain differences in these very limited studies.

Iron-carbohydrate complex showed a low order of toxicity in 3 species of laboratory animals. Studies in mice indicate that iron-carbohydrate complex is less toxic than ferrous sulfate, ferrous gluconate, ferrous fumarate, ferroglycine sulfate complex, ferric choline citrate and iron polysaccharide complex by the oral and intraperitoneal routes.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Dose descriptor:

LD50

Effect level:

>= 7 460 mg/kg bw

Based on:

test mat.

95% CL:

>= 6 844 - <= 8 131

Remarks on result:

other: 7460 (6844-8131) mg/kg (= 865 Fe, as the Fe content of ferrous gluconate is 11.58 %)

Results

Table 2 - Acute Toxicities Of Iron Compounds In Rats
LD50 (19/20Confidence Limits)
	% Fe	i.g. route
Compound		No. rats	As salt	As Fe
Iron-carbohydrate complex	48.8	10	>8000	>3904
Ferrous sulfate	29.7	24	2625 (2323-2966)	780
Ferrous gluconate	11.6	24	7460 (6844-8131)	865
Ferrous fumarate	32.9	24	>7080	>2329
Ferroglycine sulfate complex	15.9	24	5590 (4454-7014)	894
Ferric choline citrate	12.0	12	>8000	960
Iron polysaccharide complex	2.0	—	—	—

Since the iron content of the compounds varies considerably comparisons were made on the basis of actual iron content.

Results of acute toxicity studies in rats are summarized in Table 2. None of the compounds are very toxic following i.g. administration to rats and evidently there are only slight differences in the lethal effects of ferrous sulfate, ferrous gluconate and ferroglycine sulfate complex. There was no significant difference between the 1- and 7-day LD₅₀ for ferrous sulfate and iron-carbohydrate complex in rats.

Iron-carbohydrate complex showed a low order of toxicity in 3 species of laboratory animals.

None of the compounds tested was less toxic than iron-carbohydrate complex by the oral route in rats or the intravenous route in dogs.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The LD50 determined (7460 mg/kg) showed the test item not to be toxic after single oral administration.
This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

This report is concerned with the animal toxicities of a new high molecular iron-carbohydrate complex compared to those of several other preparations (Weaver, 1961). For this purpose several toxicity test were conducted in different animal species. In the acute toxicity test in rats the compounds (i.e. ferrous gluconate) were administered as aqueous solutions where possible, otherwise as fine suspensions. Male (250 to 350 g) and female (150 to 250 g) Harlan-Wistar rats in mixed groups of 6 received the iron compounds i.g. in attempts to determine 24-hour LD₅₀ in this species. The animals were observed closely for several hours following injection, and the LD₅₀ and 95% confidence limits were determined at the end of 24 hours by the method of Litchfield and Wilcoxon.

Since the iron content of the compounds varies considerably comparisons were made on the basis of actual iron content. None of the compounds are very toxic following i.g. administration to rats and evidently there are only slight differences in the lethal effects of ferrous sulfate, ferrous gluconate (LD₅₀ = 7460 mg/kg) and ferroglycine sulfate complex. There was no significant difference between the 1- and 7-day LD₅₀ for ferrous sulfate and iron-carbohydrate complex in rats.

Thus, iron-carbohydrate complex showed a low order of toxicity in 3 species of laboratory animals. In addition, none of the compounds tested was less toxic than iron-carbohydrate complex by the oral route in rats.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 6

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rational for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

male

Dose descriptor:

LD50

Effect level:

>= 3 700 mg/kg bw

Remarks on result:

other: as salt ( ± S.E. of 145)

Sex:

male

Dose descriptor:

LD50

Effect level:

>= 429 mg/kg bw

Remarks on result:

other: as Fe++ ( ± S.E. of 17)

Acute Toxicity Studies In The Mouse

Oral. The acute oral toxicity data in Table 1 show that ferrous gluconate is significantly less toxic than ferrous sulfate, both in terms of the salt and of ferrous iron. The oral LD₅₀ for ferrous sulfate was found to be 1520 mg/kg compared with 3700 mg/kg of ferrous gluconate which, when expressed in terms of ferrous iron, amounts to 306 mg/kg as the sulfate and 429 mg/kg as the gluconate. These differences are statistically significant and indicate that the gluconate, in terms of ferrous iron content, is approximately 40% better tolerated than the sulfate. There were no delayed deaths with either compound following oral administration in mice.

Table 1 - Acute Toxicity Of Ferrous Sulfate (FeSO4-7H2O) Versus Ferrous Gluconate (Fe[C6H11O7]2-2H2O) In Mice
LD50 ± s.e. mg/kg
Compound	Route of Administration	No. of Animals	As Salt		As Fe^++
			24 Hours	7 Days	24 Hours	7 Days
Ferrous sulfate	I.V.	30	65 ± 4.8	51 ± 4.6	13 ± 1	10.2 ± 0.9
Ferrous gluconate	I.V.	40	114 ± 7.6	98 ± 6.8	12.5 ± 0.7	10.8 ± 0.7
Ferrous sulfate	Oral	30	1520 ± 130	1520 ± 130	306 ± 26	306 ± 26
Ferrous gluconate	Oral	60	3700 ± 145	3700 ± 145	429 ± 17	429 ± 17

Discussion

Comparison of the present acute toxicity data on ferrous gluconate and ferrous sulfate with the data available in the literature indicates some agreement and also some wide discrepancies. The present acute oral LD₅₀ values of 1520 ± 130 mg/kg for ferrous sulfate (FeSO₄*7H₂O) and 3700 ± 145 mg/kg for ferrous gluconate (Fe[C₆H₁₁O₇]₂ *2H₂O) in the mouse indicate a higher acute oral toxicity for these substances than that reported for the mouse in the literature. These variations may be due to differences in methods of administration, strain of mice, conditions of assay, and the like.

It is of interest to note that the estimated oral median lethal dose of 900 mg/kg for ferrous sulfate in children, referred to earlier, is within the limits of experimental error for the acute oral LD₅₀ values for ferrous sulfate in the mouse (1520 ± 130 mg/kg ) and the rat (1480 ± 184 mg/kg) as established in the present investigation.

Summary

The results of a direct comparison of the acute systemic and local toxicity of ferrous sulfate (FeSO₄ *7H₂O) and ferrous gluconate (Fe[C₆H₁₁O₇]₂ *2H₂O) in experimental animals may be summarized as follows:

Studies in mice indicate that the acute oral toxicity values (ferrous sulfate, 1520 ± 130 mg/kg; ferrous gluconate, 3700 ± 145 mg/ kg) were more than twenty times as large as those following acute intravenous injection. No delayed deaths of significance were observed following oral administration.

The magnitude of the acute oral toxicity values when compared with the acute intravenous figures in mice indicates a relatively low order of absorption from the intestinal tract.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

The acute toxicity of ferrous gluconate was determined in direct comparison with that of ferrous sulfate following both intravenous and oral administration in male, albino Swiss mice weighing 22 ± 2 g (Hoppe, 1955). A volume of 0.01 cc./g of body weight in aqueous solution was used for oral administration. All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The mice were fasted for 4 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The mice were observed closely for several hours following injection, and the LD₅₀ ± its standard error was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD₅₀ was recalculated at the end of the 7-day observation period.

The acute oral toxicity data show that ferrous gluconate is significantly less toxic than ferrous sulfate, both in terms of the salt and of ferrous iron. The oral LD₅₀ for ferrous sulfate was found to be 1520 mg/kg compared with 3700 mg/kg of ferrous gluconate which, when expressed in terms of ferrous iron, amounts to 306 mg/kg as the sulfate and 429 mg/kg as the gluconate. These differences are statistically significant and indicate that the gluconate, in terms of ferrous iron content, is approximately 40% better tolerated than the sulfate. There were no delayed deaths with either compound following oral administration in mice.

Studies in mice indicate that the acute oral toxicity of ferrous gluconate (3700 ± 145 mg/kg) is approximately half that of ferrous sulfate (1520 ± 130 mg/kg) in terms of absolute weights of the salts. In terms of iron, the toxicity of the two compounds shows a similar relationship. No delayed deaths of significance were observed following oral administration in either species.

The magnitude of the acute oral toxicity values when compared with the acute intravenous figures in mice indicates a relatively low order of absorption from the intestinal tract. An additional safety factor is evident from the oral studies in the cat and the dog in which the local irritant effects induce a protective emesis. These data suggest prompt, gentle gastric lavage along with supportive therapy for shock as an effective emergency measure in those cases where, for any reason, vomiting does not occur spontaneously following oral ingestion of ferrous sulfate, ferrous gluconate or other soluble iron salts.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 7

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

male

Dose descriptor:

LD50

Effect level:

4 600 mg/kg bw

Remarks on result:

other: ± S.E. of 560 (as salt, after 24 hours)

Sex:

male

Dose descriptor:

LD50

Effect level:

518 mg/kg bw

Remarks on result:

other: ± S.E. of 63 (as Fe++, after 24 hours)

Sex:

male

Dose descriptor:

LD50

Effect level:

507 mg/kg bw

Remarks on result:

other: ± S.E. of 45 (As Fe++, after 7 days)

Sex:

male

Dose descriptor:

LD50

Effect level:

4 500 mg/kg bw

Remarks on result:

other: ± S.E. of 400 (as salt, after 7 days)

Acute Oral Toxicity Studies In The Rat

The acute oral toxicity data in the rat were found to be of a similar order of magnitude as those found in the mouse as will be noted from the data in Table 2. In terms of the salts, ferrous gluconate was found to be approximately one-third as toxic as ferrous sulfate following oral administration in the rat. When compared in terms of ferrous iron, ferrous gluconate is significantly less toxic, being approximately one-half as toxic as ferrous sulfate. No delayed deaths were observed with ferrous sulfate; one delayed death was observed with ferrous gluconate.

Table 2 - Acute Toxicity Of Ferrous Sulfate (FeSO4-7H2O) Versus Ferrous Gluconate (Fe[C6H11O7]2-2H2O) In Rats
LD50 ± s.e. mg/kg
Compound	No. of -Animals	As Salt		As Fe++
		24 Hours	7 Days	24 Hours	7 Days
Ferrous sulfate	30	1480 ± 184	1480 ± 184	298 ± 37	298 ± 37
Ferrous gluconate	30	4600 ± 560	4,500 ± 400	518 ± 63	507 ± 45

Discussion

Comparison of the present acute toxicity data on ferrous gluconate and ferrous sulfate with the data available in the literature indicates some agreement and also some wide discrepancies.

Summary

The results of a direct comparison of the acute systemic and local toxicity of ferrous sulfate (FeSO₄ *7H₂O) and ferrous gluconate (Fe[C₆H₁₁O₇]₂ *2H₂O) in experimental animals may be summarized as follows: In the rat ferrous gluconate (4600 ± 560 mg/kg) was only one-third as toxic as ferrous sulfate (1480 ± 184 mg/kg) as the salt and one-half as toxic in terms of ferrous iron. No delayed deaths of significance were observed following oral administration.

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

The acute toxicity of ferrous gluconate was determined in direct comparison with that of ferrous sulfate following oral administration in male, Sprague-Dawley rats weighing 100 ± 10 g (Hoppe, 1955). A volume of 0.01 cc./g of body weight in aqueous solution was used for oral administration. All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The rats were fasted for 4 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The rats were observed closely for several hours following injection, and the LD₅₀ ± its standard error was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD₅₀ was recalculated at the end of the 7-day observation period.

The acute oral toxicity data in the rat were found to be of a similar order of magnitude as those found in the mouse. Studies indicate that the acute oral toxicity of ferrous gluconate (4600 ± 560 after 24 hours or 4500 ± 400 mg/kg after 7 days) was only one-third as toxic as ferrous sulfate (1480 ± 184 mg/kg after 24 hours or 7 days) as the salt and one-half as toxic in terms of ferrous iron. No delayed deaths were observed following oral administration of ferrous sulfate; one delayed death was observed with ferrous gluconate.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 8

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

not specified

Dose descriptor:

LD50

Effect level:

> 400 mg/kg bw

Remarks on result:

other: It was not possible to obtain mortality data by this route of administration at the dose levels employed, since emesis occurred in every cat within 15 minutes to one hour after medication.

Mortality:

no mortality occurred

Clinical signs:

emesis occurred in every cat within 15 minutes to one hour after medication. Severe diarrhea also was observed but became less evident at the higher dosages as the promptness and intensity of emesis increased.

Body weight:

no data

Gross pathology:

not performed

Other findings:

no data

Acute Oral Toxicity In The Cat

It was not possible to obtain mortality data by this route of administration at the dose levels employed, since emesis occurred in every cat within 15 minutes to one hour after medication. Severe diarrhea also was observed but became less evident at the higher dosages as the promptness and intensity of emesis increased. It was concluded from these experiments that the acute oral lethal dose of ferrous sulfate in cats was more than 200 mg/kg and more than 400 mg/kg for ferrous gluconate. The pattern of emesis was sufficiently prominent and consistent to permit the estimation of the approximate median emetic dose, AED₅₀, (the approximate dose producing emesis in 50% of the cats) as a criterion for comparing the gastric tolerance to these two compounds in cats. A summary of the emetic effects and of the incidence of diarrhea is given in Table 3. It will be noted that the dose of ferrous gluconate required to produce emesis in 50% of the cats was more than three times as large as that of ferrous sulfate. About twice as much iron in the form of ferrous gluconate was tolerated without vomiting as was tolerated in the form of the sulfate.

Table 3 - Effects Of Single Oral Dosages Of Ferrous Sulfate (FeSO4-7H2O) and Ferrous Gluconate (Fe[C6H11O7]2-2H2O) in Cats
		Emetic Effects			Diarrhea
Compound	Dose mg/kg	No. Vomited/No. Medicated	AED50 mg/kg		No. Showing Diarrhea/No. Medicated
			As Salt	As Fe++
Ferrous sulfate	25	1/4	82	16	2/4
	50	2/4	-	-	2/4
	100	1/4	-	-	0/4
	200	4/4	-	-	0/4
Ferrous gluconate	100	0/4	267	31	2/4
	200	1/4	-	-	1/4
	400	4/4	-	-	0/4

Discussion

Comparison of the present acute toxicity data on ferrous gluconate and ferrous sulfate with the data available in the literature indicates some agreement and also some wide discrepancies. In the case of the cat, a literature value of greater than 500 mg/kg of ferrous sulfate was reported. In the present study, no mortality was observed with single oral doses of ferrous sulfate up to and including 200 mg/kg. When given by repeated oral administration, however, one of two cats died at the end of the first week at a dose of 400 mg/kg of ferrous sulfate. Copious vomiting was encountered in both the cat and the dog, which tended to interfere with attempts to estimate the acute oral lethal dose of ferrous sulfate in these two species. The fact that copious and effective emesis interfered with the estimation of the acute oral lethal dose of ferrous sulfate in both the cat and dog indicates that this protective mechanism may be better developed in these two species than it is in the human. It is of interest to note that the estimated oral median lethal dose of 900 mg/kg for ferrous sulfate in children, referred to earlier, is within the limits of experimental error for the acute oral LD₅₀ values for ferrous sulfate in the mouse (1520 ± 130 mg/kg ) and the rat (1480 ± 184 mg/kg) as established in the present investigation.

Summary

The results of a direct comparison of the acute systemic and local toxicity of ferrous sulfate (FeSO₄ *7H₂O) and ferrous gluconate (Fe[C₆H₁₁O₇]₂ *2H₂O) in experimental animals may be summarized as follows:

Attempts to estimate the acute oral toxicity in cats were unsuccessful, due to intense local gastric irritation which resulted in prompt and copious vomiting. Approximately twice as much ferrous iron in the form of ferrous gluconate as ferrous sulfate was tolerated before vomiting occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

The acute toxicity of ferrous gluconate was determined in direct comparison with that of ferrous sulfate following oral administration in cats (Hoppe, 1955). Ferrous gluconate and ferrous sulfate were administered orally as a finely divided powder by capsule to cats, weighing 2 to 3 kg, in an effort to determine the acute lethal dose following oral administration.

All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The cats were fasted for 18 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The cats were observed closely for several hours following injection, and the LD₅₀ ± its standard error was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD₅₀ was recalculated at the end of the 7-day observation period.

It was not possible to obtain mortality data by this route of administration at the dose levels employed, since emesis occurred in every cat within 15 minutes to one hour after medication. Severe diarrhea also was observed but became less evident at the higher dosages as the promptness and intensity of emesis increased. It was concluded from these experiments that the acute oral lethal dose of ferrous sulfate in cats was more than 200 mg/kg and more than 400 mg/kg for ferrous gluconate. The pattern of emesis was sufficiently prominent and consistent to permit the estimation of the approximate median emetic dose, AED₅₀, (the approximate dose producing emesis in 50% of the cats) as a criterion for comparing the gastric tolerance to these two compounds in cats. It will be noted that the dose of ferrous gluconate required to produce emesis in 50% of the cats was more than three times as large as that of ferrous sulfate. About twice as much iron in the form of ferrous gluconate was tolerated without vomiting as was tolerated in the form of the sulfate.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 9

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

not specified

Dose descriptor:

LD50

Effect level:

> 3 200 mg/kg bw

Mortality:

No deaths or serious evidence of acute systemic intoxication were observed in the dogs at doses up to and including the highest dose level, 800 mg/kg of ferrous sulfate or 3200 mg/kg of ferrous gluconate.

Clinical signs:

No deaths or serious evidence of acute systemic intoxication were observed in the dogs at doses up to and including the highest dose level, 800 mg/kg of ferrous sulfate or 3200 mg/kg of ferrous gluconate. The most obvious effects produced by these two compounds were emesis and diarrhea. With ferrous gluconate, vomiting did not occur until doses of 1600 and 3200 mg/kg were reached. A watery diarrhea became apparent approximately one hour after oral administration of 800 mg/kg of ferrous gluconate. At doses of 200 and 400 mg/kg of ferrous gluconate, diarrhea developed the morning of the day following medication.

Body weight:

no data

Gross pathology:

not performed

Other findings:

no data

Acute Oral Toxicity Studies In The Dog

Six dogs, one at each dosage level, were given capsules of finely divided ferrous gluconate in amounts ranging from 100 to 3200 mg/kg Five other dogs received similar capsules of ferrous sulfate in doses from 50 to 800 mg/kg. No deaths or serious evidence of acute systemic intoxication were observed in the dogs at doses up to and including the highest dose level, 800 mg/kg of ferrous sulfate or 3200 mg/kg of ferrous gluconate. The most obvious effects produced by these two compounds were emesis and diarrhea (Table 4). Vomiting was noted in the dog receiving 50 mg/kg of ferrous sulfate but was not encountered in the others until the dose was raised to 800 mg/kg, when a prompt and vigorous emetic reaction was observed. With ferrous gluconate, vomiting did not occur until doses of 1600 and 3200 mg/kg were reached. A watery diarrhea became apparent approximately one hour after oral administration of 100 mg/kg of ferrous sulfate and 800 mg/kg of ferrous gluconate. At doses of 200 and 400 mg/kg of ferrous gluconate, diarrhea developed the morning of the day following medication. The occurrence of vomiting and diarrhea, indicative of a protective mechanism similar to that observed in the cat, interfered with the attempt to estimate the acute oral lethal dosage of these compounds in dogs.

Table 4 - Effects Of Single Oral Dosages Of Ferrous Sulfate (FeSO4-7H2O) and Ferrous Gluconate (Fe[C6H11O7]2-2H2O) in Dogs
	Done, mg./kg.
Compound	As Salt	As Fe^++	Vomiting	Diarrhea
Ferrous sulfate	50	10.0	Yes	No
	100	20.1	No	Yes at 2 hours
	200	40.2	No	Yes at 1 hour
	400	80.4	No	Yes at 1 hour
	800	160.8	Yes at 10 min.	Yes at 13 hours
Ferrous gluconate	100	11.6	No	No
	200	23.2	No	Yes at 24 hours
	400	46.4	No	Yes at 24 hours
	800	92.8	No	Yes at 11 hours
	1600	185.6	Yes at 1 hour	Yes at 2 hours
	3200	371.2	Yes at 11 hours	Yes at 1 hour

Discussion

Comparison of the present acute toxicity data on ferrous gluconate and ferrous sulfate with the data available in the literature indicates some agreement and also some wide discrepancies.

An estimated acute oral lethal dose of 800 mg/kg of ferrous sulfate for the dog has been reported. In the present study no mortality was observed following oral administration of ferrous sulfate at dosages up to and including 800 mg/kg in the dog. Copious vomiting was encountered in both the cat and the dog, which tended to interfere with attempts to estimate the acute oral lethal dose of ferrous sulfate in these two species. The fact that copious and effective emesis interfered with the estimation of the acute oral lethal dose of ferrous sulfate in both the cat and dog indicates that this protective mechanism may be better developed in these two species than it is in the human.

Summary

The results of a direct comparison of the acute systemic and local toxicity of ferrous sulfate (FeSO₄ *7H₂O) and ferrous gluconate (Fe[C₆H₁₁O₇]₂ *2H₂O) in experimental animals may be summarized as follows:

In the dog the acute oral median lethal dose was estimated to be greater than 800 mg/kg of ferrous sulfate and more than 3200 mg/kg of ferrous gluconate. No deaths or serious evidence of acute systemic intoxication were observed at these doses. The emesis and diarrhea produced by both compounds rendered attempts to estimate accurate LD₅₀ values impracticable.

The magnitude of the acute oral toxicity values when compared with the acute intravenous figures in mice indicates a relatively low order of absorption from the intestinal tract. An additional safety factor is evident from the oral studies in the cat and the dog in which the local irritant effects induce a protective emesis. These data suggest prompt, gentle gastric lavage along with supportive therapy for shock as an effective emergency measure in those cases where, for any reason, vomiting does not occur spontaneously following oral ingestion of ferrus sulfate, ferrous gluconate or other soluble iron salts.

Interpretation of results:

GHS criteria not met

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

The acute toxicity of ferrous gluconate was determined in direct comparison with that of ferrous sulfate following oral administration as a finely divided powder by capsule in mongrel dogs weighing 7 to 12 kg, in an effort to determine the acute lethal dose following oral administration (Hoppe, 1955). Five dogs for ferrous sulfate and six dogs for ferrous gluconate, one at each dosage level (50, 100, 200, 400 and 800 mg/kg for ferrous sulfate and 100, 200, 400, 800, 1600 and 3200 mg/kg for ferrous gluconate), were given capsules of finely divided ferrous gluconate in amounts ranging from 100 to 3200 mg/kg. All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The dogs were fasted for 18 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The dogs were observed closely for several hours following injection, and the LD₅₀ ± its standard error was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD₅₀ was recalculated at the end of the 7 -day observation period.

No deaths or serious evidence of acute systemic intoxication were observed in the dogs at doses up to and including the highest dose level, 800 mg/kg of ferrous sulfate or 3200 mg/kg of ferrous gluconate. The most obvious effects produced by these two compounds were emesis and diarrhea. Vomiting was noted in the dog receiving 50 mg/kg of ferrous sulfate but was not encountered in the others until the dose was raised to 800 mg/kg, when a prompt and vigorous emetic reaction was observed. With ferrous gluconate, vomiting did not occur until doses of 1600 and 3200 mg/ kg were reached. A watery diarrhea became apparent approximately one hour after oral administration of 100 mg/kg of ferrous sulfate and 800 mg/ kg of ferrous gluconate. At doses of 200 and 400 mg/kg of ferrous gluconate, diarrhea developed the morning of the day following medication. The occurrence of vomiting and diarrhea, indicative of a protective mechanism similar to that observed in the cat, interfered with the attempt to estimate the acute oral lethal dosage of these compounds in dogs.

In summary, the acute oral median lethal dose was estimated to be greater than 800 mg/kg of ferrous sulfate and more than 3200 mg/kg of ferrous gluconate. No deaths or serious evidence of acute systemic intoxication were observed at these doses. The emesis and diarrhea produced by both compounds rendered attempts to estimate accurate LD₅₀values impracticable.

The magnitude of the acute oral toxicity values when compared with the acute intravenous figures in mice indicates a relatively low order of absorption from the intestinal tract. An additional safety factor is evident from the oral studies in the cat and the dog in which the local irritant effects induce a protective emesis.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 10

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

from Public Assessment Report - Scientific discussion

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

not specified

Dose descriptor:

LD50

Effect level:

4 600 mg/kg bw

Ferro gluconate has been extensively used for decades with an established and well-known efficacy and safety. The proposed dose and schedule is typical and it does not need further documentation. This “pivotal study” is a very small study and it is difficult to conclude much from these sparse data. However, it is indicated that treatment with ferro gluconate is as good as ferro sulphate no matter the amount of ascorbic acid added to each tablet. In view of the well-knowness of iron therapy there is no need for further efficacy data.

There are no safety concerns related to the use of this product.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

This assessment report concerns Fexin effervescent tablets which contains Iron (II) as Ferrous gluconate and were approved through DCP (DK/H/1074/001/DC) on 3 October 2007 with Denmark as RMS. Based on the review of the data on quality, safety and efficacy, it was considered that the application for Fexin indicated for:

• Iron deficiency anaemia

• Iron deficiency

• Prophylactic use in pregnancy and for blood donors could be approved.

The application concerns Iron (II) as Ferrous Gluconate effervescent tablets each containing 80.5 mg Iron (II).

In this report a LD50 value of 4600 mg/kg for rats is mentionned, however, no further reference is given.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 11

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

from Public Assessment Report - Scientific discussion

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

not specified

Dose descriptor:

LD50

Effect level:

> 2 200 mg/kg bw

Clinical signs:

The principal findings included gastro-intestinal inflammation and ulceration in the rat and vomiting in the dog.

Ferro gluconate has been extensively used for decades with an established and well-known efficacy and safety. The proposed dose and schedule is typical and it does not need further documentation. This “pivotal study” is a very small study and it is difficult to conclude much from these sparse data. However, it is indicated that treatment with ferro gluconate is as good as ferro sulphate no matter the amount of ascorbic acid added to each tablet. In view of the well-knowness of iron therapy there is no need for further efficacy data.

There are no safety concerns related to the use of this product.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

This assessment report concerns Fexin effervescent tablets which contains Iron (II) as Ferrous gluconate and were approved through DCP (DK/H/1074/001/DC) on 3 October 2007 with Denmark as RMS. Based on the review of the data on quality, safety and efficacy, it was considered that the application for Fexin indicated for:

• Iron deficiency anaemia

• Iron deficiency

• Prophylactic use in pregnancy and for blood donors could be approved.

The application concerns Iron (II) as Ferrous Gluconate effervescent tablets each containing 80.5 mg Iron (II).

In this report a LD50 value of > 2200 mg/kg for dogs is mentionned, however, no further reference is given.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 12

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

World Health Organizazion - Technical Report Series No. 576 - Evaluation Of Certain Food Additives - Nineteenth Report of the Joint FAO/WHO Expert Committee on Food Additives

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the source and the target substance and the expected identical behaviour in the human or animal body. This is based on the fact, that they both are metal complexes consisting of iron and sugar-like carbohydrates that are believed to share the same absorption, distribution and metabolic pathways.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity, 2) the same behaviour in the acidic environment in the stomach and proximal duodenum (dissociation), 3) the same oxidation status of the iron ion after absorption, 4) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 5) its identical limited elimination mechanisms.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Ferrous gluconate, CAS 299-29-6,
SMILES [Fe+2].O[C@H]([C@@H](O)C([O-])=O)[C@H](O)[C@H](O)CO.[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO,
MW 446.14 g/mol
The molecular formula is considered to be C12H22FeO14. It is used to replete and maintain the total body content of iron.
The purity, when specified, is given in terms of iron content, which is ca .11.7 % (11.58 % / 11.6 %/ 11.7 %)

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Ferrous gluconate or Iron(II) gluconate (source) and Sodium Iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - contain the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length. In case of ferrous gluconate two gluconic acid-chains (C6H11O7-) are involved and in case of iron glucoheptonate it is C7H10O8.
After oral intake of these substances the low pH in the upper GI tract will provoke dissociation releasing gluconic acid / glucoheptonic acid and ferrous and ferric iron, respectively. Therefore, at low pH values both substances are not able to participate in complexation of metal cations (Alekseev et al., 1998).
This dissociation has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998).
In the posterior parts of the GI tract, in the small intestines, where pH raises, new complexes can be formed, impacting any additional absorption of both substances again in a similar way. This however is not of great importance as the major absorption of iron takes place in the upper GI tract, where the pH values are low.
The released free gluconate or glucoheptonate anions, however, can further sequester luminal or mucosal metal affecting the absorption. The impact of this on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13.). Therefore gluconate-metal complexes are used for food fortification. Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13.).
All this shows that the metal cation originated from the gluconate /glucoheptonate complexes is subjected to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Substantiating these facts, it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.Moreover, lactonisation occurs at low pH values (i.e. observed under pH 3.8 in case of Ca gluconate complexes) that would hinder complexation (Pallagi et al., 2010).
The ferric iron of sodium ferric glucoheptonate needs to be transformed into the soluble ferrous iron before being absorbed (Andrews, 2000). The reduction of ferric iron is aided by the acidic environment of the stomach and the proximal duodenum (enhancing in the first place solubility), dietary components like ascorbic acid and duodenal cytochrome b (Dcytb) catalysing duodenal ferric reductase activity (Atanasova, 2005). Thus the metal ion is expected to be absorbed similarly in both cases, despite the different origin oxidation status.
Generally, the extent of iron absorption depends on body needs and is highly regulated (EFSA, 2006; Candela et al., 1984). Subsequently, after absorption, the sugar residues, which differ by one methyl rest, will both be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to gluconate or glucoheptonate moiety up to considerable amounts.
Taken together, both substances are expected to have an identical toxicodynamic and toxicological behaviour, which is based on the fact that these similar structures are metabolised by the same pathways, leading to the same substances.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file.

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute toxicity

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Dose descriptor:

other: Acceptable daily intake (ADI)

Remarks on result:

other: ADI not specified - the contribution from ferrous gluconate to the total dietary gluconic acid intake from all sources should be included in the ADI for gluconic acid.

Ferrous gluconate

This compound was evaluated by the Committee not in relation to its use as a nutritional supplement but as a colouring adjunct. It was given an " ADI not specified ", with the proviso that the contribution from ferrous gluconate to the total dietary gluconic acid intake from all sources should be included in the ADI for gluconic acid. Specifications were prepared at the eighteenth meeting of the Committee.

Conclusions:

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Executive summary:

Ferrous gluconate

This compound was evaluated by the Committee not in relation to its use as a nutritional supplement but as a colouring adjunct. It was given an " ADI not specified ", with the proviso that the contribution from ferrous gluconate to the total dietary gluconic acid intake from all sources should be included in the ADI for gluconic acid. Specifications were prepared at the eighteenth meeting of the Committee.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not acutely toxic according to 1272/2008/EC.

Reference 13

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the organic part of the source and the target substance and their expected identical behaviour in the human or animal body.
The target substance – as one member in the group of the chelate complexes - is known to dissociate in the upper GI tract at low pH values into the metal ion and the glucoheptonic acid anion.
The read-across substances gluconic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate and calcium gluconate similarly release gluconate anions which are as well sugar-like compounds and are metabolised by the same metabolic pathways as glucoheptonate anion, and are indeed intermediate metabolites, which are partly build when the glucoheptonate anion is broken down.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity and 2) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 3) their identical excretion mechanisms.

Therefore, by using the toxicity profile of gluconic acid, glucono-delta-lactone, sodium, potassium and calcium gluconate, the absence of toxicity of the organic moiety of the target molecule - glucoheptonate anion - can be confirmed.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Gluconates and derivatives
D-Gluconic acid, CAS 526-95-4;
SMILES: O=C(O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 196.16 g/mol
The molecular formula is C6H12O7
Purity (%) of Gluconic acid 50% solution: 49-52% (OECD SIDS, 2004)

Glucono-delta-lactone CAS 90-80-2
SMILES: C([C@@H]1[C@H]([C@@H]([C@H](C(=O)O1)O)O)O)O
MW 178.14 g/mol
The molecular formula is C6H10O6
Purity (%) of Glucono-delta-lactone: 99-101% (OECD SIDS, 2004)

Sodium D-gluconate CAS 527-07-1
SMILES: [Na+].[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 218.14 g/mol
The molecular formula is C6H11NaO7
Purity (%) of Sodium gluconate: 98-102% (OECD SIDS, 2004)

Calcium D-gluconate CAS-299-28-5; CAS 18016-24-5
SMILES: [Ca+2].[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.[O-C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 430.373 g/mol
The molecular formula is C12H22CaO14
Purity (%) of Calcium gluconate: 98-104% (OECD SIDS, 2004)

Potassium D-gluconate CAS 299-27-4)
SMILES: [K+].[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 234.246 g/mol
The molecular formula is C6H11KO7
Purity (%) of Potassium gluconate: 97-103% (OECD SIDS, 2004)

Gluconic acid and its derivatives glucono-delta-lactone, sodium gluconate, calcium gluconate and potassium gluconate and sodium iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - are built of the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length (gluconic acid-chains (C6H11O7-) and glucoheptonate C7H10O8).

Target Chemical:
Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION

Gluconates and their derivatives all belong to the aldonic acids (Escandar et al., 1992; Frutos et al., 1998) because they are derivatives of aldoses, sugars that contain one aldehyde group, which is oxidised to carboxylic group (Berg et al., 2007). They form lactone form, a ring structure the same as in the sugar’s cyclic hemiacetal form (Berg et al., 2007).
The dissociation of metal gluconate and glucoheptonate complexes at low pH values in the upper GI tract has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998). Consequently, the metal ion and the gluconic or glucoheptonic acid anion will be subject to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Considering this dissociation, the information that it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body is a very useful information. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.
The released free gluconate or glucoheptonate anions, however, can in the posterior parts of the GI tract – in the small intestines, where pH raises - further sequester luminal or mucosal metal affecting the absorption. The impact of the released free gluconate or glucoheptonate anions on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13). Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13).

After absorption, the above mentioned sugar residues, will all be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to the gluconate or glucoheptonate moiety up to considerable amounts.

In conclusion, gluconate and glucoheptonate anions form complexes with metals of the same geometry and stoichiometry. The same functional groups of the ligand are involved in the formation of coordinative bonds. Identical behaviour of gluconates and glucoheptonates depending on pH, molar concentration of metal: ligand components and physical state is verified by analytical methods. Based on the structural similarity of gluconates and glucoheptonates, their functional groups, composition, toxicodynamic, toxicokinetic, and toxicological behaviour, one can expect a very similar behaviour of these substances in living organisms.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute oral toxicity.

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks:

sodium gluconate

Remarks on result:

other: for rats and dogs (Mochizuki, 1995; Okamoto, 1995).

Sex:

not specified

Dose descriptor:

LD50

Effect level:

6 060 mg/kg bw

Based on:

test mat.

Remarks:

potassium gluconate

Remarks on result:

other: for rats (TNO, 1978)

Mortality:

No mortality was observed (Mochizuki, 1995; Okamoto, 1995). One animal died in the 5190 mg/kg bw group and four animals in the 6210 mg/kg bw group. Deaths occurred between 5 and 21 hours after treatment. Survivors recovered gradually (TNO, 1978).

Clinical signs:

Soft faeces and diarrhoea, seen in one male and three females at 2000 mg/kg bw, were the only clinical effects observed 2-3 h after treatment (Mochizuki, 1995).

Body weight:

The body weights of treated rats were comparable to those of controls (Mochizuki, 1995).

Gross pathology:

No gross abnormalities were observed at necropsy (Mochizuki, 1995).

Other findings:

The minimum lethal dose was > 2000 mg/kg bw, although a transient, initial laxative effect was observed in rats at doses > 1000 mg/kg bw (Mochizuki, 1995).

Data on acute oral toxicity for sodium gluconate in rat (Mochizuki, M, Bozo Research Center 1995) (doses: 500, 1000, 2000 mg/kg) and dog (Okamoto M., 1995) (doses: 1000 and 2000 mg/kg) fed by gavage showed no death at any dose, hence the minimum lethal dose was estimated > 2000 mg/kg for both species.

Rats were fed by gavage 3000, 3600, 4320, 5190, 6210 mg/kg bw (30% (w/v) aqueous solution) potassium gluconate and were observed for signs of toxicity during a 14-day period. One animal died in the 5190 mg/kg bw group and four animals in the 6210 mg/kg bw group. Deaths occurred between 5 and 21 hours after treatment. Survivors recovered gradually. The LD₅₀ was calculated (according to the method of Weil) to be 6060 mg/kg bw. However, the effects that were observed occurred at doses that exceed the accepted limit dose of 5000 mg/kg bw and the LD₅₀ may be related to high dosing (TNO, 1978). No relevant oral toxicity data were found in the literature for the other substances of the category. In conclusion, studies with sodium gluconate in the rat and dog report LD₅₀ values > 2000 mg/kg bw for both species. A gavage study with potassium gluconate and rats reported an LD₅₀ of 6060 mg/kg bw.

Interpretation of results:

practically nontoxic

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Oral LD50 > 2000 mg/kg bw was established for rats and dogs for gluconates.
These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not toxic after oral intake is important.

Executive summary:

Data on acute oral toxicity for sodium gluconate in rat (Mochizuki, M, Bozo Research Center 1995) (doses: 500, 1000, 2000 mg/kg) and dog (Okamoto M., 1995) (doses: 1000 and 2000 mg/kg) fed by gavage showed no death at any dose, hence the minimum lethal dose was estimated > 2000 mg/kg for both species.

Rats were fed by gavage 3000, 3600, 4320, 5190, 6210 mg/kg bw (30% (w/v) aqueous solution) potassium gluconate and were observed for signs of toxicity during a 14-day period. One animal died in the 5190 mg/kg bw group and four animals in the 6210 mg/kg bw group. Deaths occurred between 5 and 21 hours after treatment. Survivors recovered gradually. The LD50 was calculated (according to the method of Weil) to be 6060 mg/kg bw. However, the effects that were observed occurred at doses that exceed the accepted limit dose of 5000 mg/kg bw and the LD₅₀ may be related to high dosing (TNO, 1978). No relevant oral toxicity data were found in the literature for the other substances of the category. Conclusion Studies with sodium gluconate in the rat and dog report LD₅₀ values > 2000 mg/kg bw for both species. A gavage study with potassium gluconate and rats reported an LD50 of 6060 mg/kg bw.

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not toxic after oral intake is important.

Reference 14

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the analogue approach is the high structural similarity between the organic part of the source and the target substance and their expected identical behaviour in the human or animal body.
The target substance – as one member in the group of the chelate complexes - is known to dissociate in the upper GI tract at low pH values into the metal ion and the glucoheptonic acid anion.
The read-across substances gluconic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate and calcium gluconate similarly release gluconate anions which are as well sugar-like compounds and are metabolised by the same metabolic pathways as glucoheptonate anion, and are indeed intermediate metabolites, which are partly build when the glucoheptonate anion is broken down.
In respect to acute oral toxicity the source and the target substances are expected to bear the same toxicity potential based on their 1) structural similarity and 2) the same metabolic fate of gluconic acid and glucoheptonic acid and finally 3) their identical excretion mechanisms.

Therefore, by using the toxicity profile of gluconic acid, glucono-delta-lactone, sodium, potassium and calcium gluconate, the absence of toxicity of the organic moiety of the target molecule - glucoheptonate anion - can be confirmed.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Gluconates and derivatives
D-Gluconic acid, CAS 526-95-4;
SMILES: O=C(O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 196.16 g/mol
The molecular formula is C6H12O7
Purity (%) of Gluconic acid 50% solution: 49-52% (OECD SIDS, 2004)

Glucono-delta-lactone CAS 90-80-2
SMILES: C([C@@H]1[C@H]([C@@H]([C@H](C(=O)O1)O)O)O)O
MW 178.14 g/mol
The molecular formula is C6H10O6
Purity (%) of Glucono-delta-lactone: 99-101% (OECD SIDS, 2004)

Sodium D-gluconate CAS 527-07-1
SMILES: [Na+].[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 218.14 g/mol
The molecular formula is C6H11NaO7
Purity (%) of Sodium gluconate: 98-102% (OECD SIDS, 2004)

Calcium D-gluconate CAS-299-28-5; CAS 18016-24-5
SMILES: [Ca+2].[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.[O-C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 430.373 g/mol
The molecular formula is C12H22CaO14
Purity (%) of Calcium gluconate: 98-104% (OECD SIDS, 2004)

Potassium D-gluconate CAS 299-27-4)
SMILES: [K+].[O-]C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO
MW 234.246 g/mol
The molecular formula is C6H11KO7
Purity (%) of Potassium gluconate: 97-103% (OECD SIDS, 2004)

Gluconic acid and its derivatives glucono-delta-lactone, sodium gluconate, calcium gluconate and potassium gluconate and sodium iron glucoheptonate complex (HGA:Fe-1:1) (target) are structurally very similar. Both - the source and the target substance - are built of the same types of hydrocarbon constituents (sugar residues), which are only variable in carbon chain length (gluconic acid-chains (C6H11O7-) and glucoheptonate C7H10O8).

Target Chemical:
Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).
Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the acute toxicity of the target substance to a significant degree, since ammonium sulfate is of relatively low acute toxicity (LD50, oral, rat: 2000 - 4250 mg/kg bw) (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION

Gluconates and their derivatives all belong to the aldonic acids (Escandar et al., 1992; Frutos et al., 1998) because they are derivatives of aldoses, sugars that contain one aldehyde group, which is oxidised to carboxylic group (Berg et al., 2007). They form lactone form, a ring structure the same as in the sugar’s cyclic hemiacetal form (Berg et al., 2007).
The dissociation of metal gluconate and glucoheptonate complexes at low pH values in the upper GI tract has been confirmed in a lot of investigations, which show gluconate and glucoheptonate complexes to be more stable at alkaline conditions, while the complexes were not stable enough to be detected at acidic conditions (Escandar et al., 1996; Sawyer, 1964; Gyurcsik and Nagy, 2000; Alekseev et al., 1998). Consequently, the metal ion and the gluconic or glucoheptonic acid anion will be subject to a more or less independent fate of absorption into the systemic circulation – independent from the organic moiety and will underlie normal physiological pathways responsible for metal uptake.
Considering this dissociation, the information that it has generally been shown by a substantial body of evidence, that the toxicity profiles of chelate compounds in general depend mainly on metal ion, its affinity to this metal, and their ability to supply or to sequester it from the body is a very useful information. In the OECD SIDS report is mentioned “Evidence from the reviewed literature suggests that the eventual toxicity of the gluconate salts would be attributable to the cation rather than to the gluconate moiety of these substances. Acute toxicity responses to the various gluconate salts are comparable with other salts of the same metals and long-term toxicities seem related to the tissue deposition of these metals. Because toxicological effects of these gluconates appear to be related to their cationic components, safe and acceptable levels in foods are limited only by the nature of the specific cations…(Life Science Research Office, 1978, cited in OECD SIDS (2004))”.
The released free gluconate or glucoheptonate anions, however, can in the posterior parts of the GI tract – in the small intestines, where pH raises - further sequester luminal or mucosal metal affecting the absorption. The impact of the released free gluconate or glucoheptonate anions on absorption of metals has been addressed by a lot of investigations, which showed that gluconate complexes actually enhanced absorption of metals increasing their bioavailability. Absorption of the nutrient metals was higher from gluconates than from the soluble inorganic compounds (i.e. Cousins, 1985, please refer to the detailed read-across statement attached in IUCLID section 13). Absorption of nutrient metals from glucoheptonate complexes seems to be equal to that from gluconate complexes (i.e. Durisova et al., 1985, please refer to the detailed read-across statement attached in IUCLID section 13).

After absorption, the above mentioned sugar residues, will all be metabolised by the pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) for the synthesis of the same 5-carbon sugars. In fact, no toxicity is attributed to the gluconate or glucoheptonate moiety up to considerable amounts.

In conclusion, gluconate and glucoheptonate anions form complexes with metals of the same geometry and stoichiometry. The same functional groups of the ligand are involved in the formation of coordinative bonds. Identical behaviour of gluconates and glucoheptonates depending on pH, molar concentration of metal: ligand components and physical state is verified by analytical methods. Based on the structural similarity of gluconates and glucoheptonates, their functional groups, composition, toxicodynamic, toxicokinetic, and toxicological behaviour, one can expect a very similar behaviour of these substances in living organisms.

Please refer also to the extended-read-across statement attached in section 13 of this IUCLID file

4. DATA MATRIX
The table attached in section 13 shows the available data relevant to justify the read-across from the source to the target chemical for the endpoint acute oral toxicity.

Reason / purpose for cross-reference:

read-across source

Sex:

not specified

Dose descriptor:

other: Estimate of acceptable daily intakes for man

Effect level:

0 - 15 mg/kg bw

Based on:

test mat.

Remarks on result:

other: unconditional acceptance (established at the tenth meeting)

Sex:

not specified

Dose descriptor:

other: Estimate of acceptable daily intakes for man

Effect level:

15 - 50 mg/kg bw

Based on:

test mat.

Remarks on result:

other: conditional acceptance (established at the tenth meeting)

Sex:

not specified

Dose descriptor:

other: Acceptable Daily Intake (ADI) for man

Effect level:

other: not specified

Based on:

test mat.

Remarks on result:

other: At its thirtieth meeting, the Committee changed the ADI for glucono-delta-lactone to an ADI 'not specified'

Sex:

female

Dose descriptor:

LDLo

Effect level:

> 10 000 other: ppm in feed

Based on:

test mat.

Remarks:

Glucono-delta-lactone

Remarks on result:

other: in rats

Sex:

not specified

Dose descriptor:

LD50

Effect level:

5 940 mg/kg bw

Based on:

test mat.

Remarks:

Glucono-delta-lactone

Remarks on result:

other: in rats

Sex:

not specified

Dose descriptor:

LD50

Effect level:

6 800 mg/kg bw

Based on:

test mat.

Remarks:

Glucono-delta-lactone

Remarks on result:

other: in mouse

Sex:

not specified

Dose descriptor:

LD50

Effect level:

7 850 mg/kg bw

Based on:

test mat.

Remarks:

Glucono-delta-lactone

Remarks on result:

other: in rabbits

Sex:

not specified

Dose descriptor:

LD50

Effect level:

5 600 mg/kg bw

Based on:

test mat.

Remarks:

Glucono-delta-lactone

Remarks on result:

other: hamster

Humans

When three men were given 10 g (167 mg/kg) of glucono-delta-lactone orally as a 10 per cent. solution, the amounts recovered in the urine in 7 hours represented 7.7-15 per cent. of the dose. No pathological urine constituents were noted. When 5 g (84 mg/kg) was given orally none was recovered in the urine. The largest dose given to man was 30 g (500 mg/kg) (Chenoweth et al., 1941).

The administration for 3-6 days of large oral doses (5-10 g/day) of gluconic acid to five normal humans did not produce any renal changes, as by the absence of blood, protein, casts and sugar in the urine (Chenoweth et al., 1941).

Short-term studies

Rat. Groups of 20 male and 20 female rats were fed gluconic acid (as glucono-delta-lactone) for 26 weeks at levels of 0 and 10 000 ppm in the diet without ill effects or demonstrable changes in the main organs on microscopic examination (Harper & Gaunt, 1962).

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Glucono-delta-lactone and gluconic acid are not toxic to animals and humans when given at very high dose levels.
These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that glucono-delta-lactone and gluconic acid are not toxic after oral intake is important.

Executive summary:

Consideration of glucono-delta-lactone and gluconic acid is based mainly on the metabolic evidence that these compounds are intermediates in a normal pathway of glucose metabolism in mammalian species. There is also considerable experience with the comparatively low toxicity of gluconate to man and animals.

Glucono-delta-lactone and gluconic acid are not toxic to animals and humans when given at very high dose levels (> 2000 mg/kg bw).

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that glucono-delta-lactone and gluconic acid are not toxic after oral intake is important.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 735 mg/kg bw

Quality of whole database:

The quality of the whole database is considered sufficient for estimation of acute toxicity potential, because of the multipicity of available data for the different source substances.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Each day two animals were exposed to Fe2O3 test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four repetitions (days) with a new set of animals each day were carried out. At the end of each five-hour exposure, two animals from each group had in-vivo chemiluminescence IVCL (Boveris et al. 1980). The IVCL technique is a highly sensitive method for identifying cardiopulmonary responses to inhaled ENMs under relatively small doses and acute exposures.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratories (Rensselae, NY)
- Weight at study initiation: 200-250 g
- Housing: housed, and managed according to the NIH guidelines for the care and use of laboratory animals
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
not specified

Male Sprague-Dawley rats (200-250 g) were obtained from Taconic Laboratories (Rensselae, NY), housed, and managed according to the NIH guidelines for the care and use of laboratory animals. Upon arrival, animals were assigned a unique identification number, which determined the exposure date and exposure group (Aerosol or Filtered Air) for the animal. Rats were allowed to acclimate to the animal facility for 4-5 days prior to start of experiments. The Harvard Medical Area’s Animal Use Committee approved the animal protocols used in this study. Each day two animals were exposed to Fe2O3 test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four repetitions (days) with a new set of animals each day were carried out. At the end of each five-hour exposure, two animals from each group had IVCL (Boveris et al. 1980).

Route of administration:

inhalation

Details on inhalation exposure:

Please refer to "Any other information on materials and methods"

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Total particle number concentration (CT) of the test ENM aerosol containing Fe2O3 nanoparticles was monitored continuously. It remains constant at ca. 2-3·105 #/cm3. Every 25 minutes there was a 5 minutes break during the system was filled up (gray area).

Duration of exposure:

5 h

Remarks on duration:

Each day two animals were exposed to Fe2O3 test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four repetitions (days) with a new set of animals each day were carried out.

Concentrations:

Total particle number concentration (CT) of the test ENM aerosol containing Fe2O3 nanoparticles remains constant at approximately 2-3·105 #/cm³. Every 25 minutes there was a 5 minutes break during the system was filled up (gray area).

No. of animals per sex per dose:

Each day two animals were exposed to aerosol and two animals to filtered air. Four repetitions (days) with a new set of animals each day were carried out.

Control animals:

yes

Details on study design:

Please refer to "Any other information on materials and methods"

Statistics:

not specified

Remarks on result:

other: exposures to inhaled nanostructured Fe2O3 can cause both pulmonary and cardiovascular effects.

Remarks:

test concentration: 2-3·10E5 particles/cm³ (this would correspond to approximately to 100-200 μg/m³)

Mortality:

no data

Clinical signs:

other: At the end of each five-hour exposure, two animals from each group showed an in-vivo chemiluminescence (IVCL) difference (counts per second-cps/ cm²) of lungs and hearts, which corresponds to the relative reactive oxygen species (ROS) concentration.

Body weight:

no data

Gross pathology:

no data

Other findings:

no data

Here, a novel technique is presented which is suitable for both ENM in-vivo inhalation and in-vitro toxicological characterization studies. The ability of this technique to generate a variety of industry-relevant, property-controlled exposure atmospheres for inhalation studies was systematically investigated. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENM in intact animal models was also demonstrated in an in-vivo study involving Sprague-Dawley rats, using freshly generated nano-iron oxide (Fe₂O₃) as a test aerosol. Both pulmonary and systemic toxicity was demonstrated

using in-vivo chemiluminescence of heart and lung. This novel platform will make it possible for toxicologists to link physico-chemical properties of inhaled ENMs to biological outcomes and help the industry to develop safer ENM.

RESULTS

Performance characterization experiments

Effect of x/y ratio on both the aerosol size (airborne phase) and primary particle size

Figure 2d show the effect of x/y ratio on both the primary particle and the mobility diameter of Fe₂O₃. Figure 2c also shows the XRD patterns of the ex-situ collected and characterized nanoparticles as a function of the x/y ratio. This indicates that - in agreement with literature - larger Fe₂O₃ particles are formed with higher x/y ratio. This is due to their larger residence time in higher temperature zones that result in larger crystals. It is worth pointing out that the XRD-estimated average crystal particle sizes (filled triangles, Figure 2d) are consistent with their average primary particle sizes (open triangles, Figure 2d), with the latter having slightly larger values indicating polycrystalline or aggregated nanoparticles. The modal mobility diameter for the Fe₂O₃ aerosol (Figure 2d, circles, left axis) is not significantly affected by the x/y ratio, as for most ratios it is fairly constant. Furthermore, as it is shown above in the case of SiO₂, there is a difference between mobility diameter (airborne phase) and the primary particle diameter (nanopowder form).

ENM surface modification

The surface of ENMs can be modified in order to add desired attributes such as dispersibility or antibacterial activity. It may also be a useful concept for the formulation of safer ENM. Recently, it was shown that doping ZnO nanoparticles with Fe can reduce the release of Zn ions. This indicates that VENGES has the ability to generate surface modified nanoparticles while maintaining the intended size distribution. This will be useful when studying the comparative toxicity of surface modified nanoparticles in-vivo. By controlling the size, they can influence control the particle deposition in the lungs. In addition, ENMs can also be used as a “vehicle” for delivery of drugs in-vivo.

Pulmonary and cardiovascular effects of inhaled nanostructured Fe₂O₃ using the VENGES platform

The concentration levels remained fairly constant for the whole animal exposure (the 5 minute stopswere necessary in order to replace the syringe with liquid precursor). For this exposure scenario, VENGES was tuned to generate a test aerosol of a total number concentration of 2-3·10⁵ particles/cm³ (this would correspond to approximately to 100-200 μg/m³), approximately 10-20 times higher concentration of indoor conditions (10 μg/m³). The temperature, relative humidity, CO₂, CO and NO₂ concentrations of the test aerosol were identical to the room conditions, ensuring no interference with the in-vivo toxicological results.

Discussion

A promising technological platform suitable for both in-vitro and in-vivo toxicological characterization of engineered nanomaterials, with emphasis on the cardiovascular and pulmonary effects of inhaled ENM, is presented in this study. ENM are produced continuously in the gas phase allowing their continuous transfer to inhalation chambers, with minimal alterations in their state of agglomeration. Defining properties of the generated aerosols (i.e. primary and aerosol particle size, concentration, shape, state of agglomeration, surface chemistry) can be easily modified by adjusting simple process parameters allowing for both in-vitro and in-vivo investigations of toxicity. The ability of the developed technique to generate a variety of industry relevant, property controlled exposure atmospheres for inhalation studies was systematically investigated and documented in the previous section. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENM in intact animal models was also demonstrated here using the highly sensitive IVCL assay.

IVCL measures the reactive oxygen species generation (ROS). ROS and free radical generation is considered one of the primary mechanisms of nanoparticle toxicity; it was shown in many ambient particle health effect studies that ROS generation may result in oxidative stress, inflammation, and damage to proteins, membranes and DNA. ROS generation has been also found in many ENMs including carbon based ENMs (fullerenes, carbon nanotubes) and metal oxides. The results indicate that moderate acute exposures to inhaled nanostructured Fe₂O₃ can generate ROS and oxidative stress and cause both pulmonary and cardiovascular effects. The IVCL measurements in the lungs of the exposed animals were about 60 times higher than for the unexposed animals, indicating that the Fe₂O₃ test aerosol increased ROS in the lungs. This oxidative stress was also present in the heart of the animals showing that the inhalation of ENM influences not only the respiratory but also the cardiovascular system with an 11-fold increase in the chemiluminescence of the heart. This substantial effect was found with a moderate mass exposure concentration of 200 μg/m³, a concentration approximately 20 times the fine particle concentration of room air. The substantial IVCL response observed in this study, indicates that these particles reached deep in the lung and evoked a toxicological effect. The increased IVCL response of the heart may indicate a direct effect on the heart (Godleski 2006) but also may be a manifestation of indirect effects via the autonomic nervous system. It should be noted, however, that the proposed platform is not only limited to the evaluation of pulmonary and cardiovascular effects. It can also be used to assess other biological outcomes related to inhaled ENM and it can be a powerful tool to understand the link between certain ENM properties and their bioavailability and toxicity.

Conclusions

In conclusion, this novel approach enables scientists and laboratory personel to generate industry-relevant, property controlled ENM exposure atmospheres suitable for inhalation toxicological studies and assess the link between ENM physico-chemical properties and specific biological outcomes. In addition, the documented in the study ability of the technique to alter in-situ ENM surface properties can be one of the ways to further explore the formulation of safer ENM. Furthermore, this technological platform can be a powerful tool for validation of in-vitro screening assays with adverse biological effects in intact animals, an important element of the strategy recently proposed by the National Nanotechnology Initiative (NNI) on Environmental Health and Safety of Engineered nanomaterials. Its future use will help to assess the cardiovascular, pulmonary and other toxicological effects of inhaled ENM and improve the understanding on the central hypothesis that physical and chemical characteristics of ENM determine their bioavailability, redistribution, and toxicity in the lungs and elsewhere.

Executive summary:

A novel method is presented which is suitable for assessing in-vivo the link between the physicochemical properties of engineered nanomaterials (ENMs) and their biological outcomes. The ability of the technique to generate a variety of industry-relevant, property-controlled ENM exposure atmospheres for inhalation studies was systematically investigated. The suitability of the technique to characterize the pulmonary and cardiovascular effects of inhaled ENMs in intact animal models is also demonstrated using in-vivo chemiluminescence (IVCL). The IVCL technique is a highly sensitive method for identifying cardiopulmonary responses to inhaled ENMs under relatively small doses and acute exposures.

Each day two animals were exposed to Fe₂O₃test aerosol and two animals to filtered air (sham) in individual exposure chambers for 5 hours. The test concentration was 2-3·10⁵particles/cm³ (this would correspond to approximately to 100-200 μg/m³). The primary particle size for Fe2O3 was controlled from 4 to 25 nm, while the corresponding agglomerate mobility diameter of the aerosol was also controlled and varied from 40 to 120 nm.

Four repetitions (days) with a new set of animals each day were carried out.

It is shown that moderate and acute exposures to inhaled nanostructured Fe₂O₃ can cause both pulmonary and cardiovascular effects.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the present study, the effects of nasal sprays of Fe2O3 (-30 nm) and ZnO (-20 nm) nanoparticles were examined in rats to identify the nature of acute inhalation toxicity.

GLP compliance:

not specified

Specific details on test material used for the study:

Fe2O3 (ca. 30 nm) and ZnO (ca. 20 nm) nanoparticles were obtained from the Institute of High Energy Physics Chinese Academy of Sciences. The sizes of the nanoparticles were confirmed by atomic force microscopy (AFM, Shimadzu, Japan). The purities of both nanoparticie preparations were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7, Thermo Electron Co.). X-ray diffraction (XRD, MSAL-XD2, China) was used to characterize the structural fingerprints of the nanoparticles.

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vitalriver Experimental Animal Technology Co. Ltd (Beijing, China).
- Females (if applicable) nulliparous and non-pregnant: [yes/no] - no as only male Wistar rats are used in the study
- Age at study initiation: adult
- Weight at study initiation: 140-160 g
- Fasting period before study: not specified
- Housing: kept in plastic cages
- Diet (e.g. ad libitum): a commercial pellet diet and allowed to access ad libitum
- Water (e.g. ad libitum): deionized water ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±2 °C
- Humidity (%): 50-70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 h light/dark rhythm

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

other: nasal sprays

Vehicle:

not specified

Mass median aerodynamic diameter (MMAD):

ca. 30 nm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
- Exposure apparatus: sprayed directly into both nasal passages using a dry powder sprayer

Average size of Fe2O3 was confirmed to be 30 ± 5 nm.

VEHICLE
- Composition of vehicle (if applicable): air
- Concentration of test material in vehicle (if applicable): not specified
- Justification of choice of vehicle: no data
- Lot/batch no. (if required): not available
- Purity: not specified

ZnO and Fe2O3 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer. Nanoparticles were placed into a sprayer and then transferred to the rat with a length of flextubing inserted into the rat nostrils. The daily administration doses were 8.5 mg/kg body weight for Fe2O3 and 2.5 mg/kg body weight for ZnO nanoparticles.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Average size of Fe2O3 was confirmed to be 30 ± 5 nm. The shape of Fe2O3 nanoparticles was spherical. The purity of both nanoparticie preparations was more than 99%. Sodium & chlorine content was < 0.001%

Remarks on duration:

ZnO and Fe203 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer

Concentrations:

8.5 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

yes

Remarks:

The rats in the control group were nasally sprayed with ambient air.

Details on study design:

ZnO and Fe2O3 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer. Nanoparticles were placed into a sprayer and then transferred to the rat with a length of flextubing inserted into the rat nostrils. The daily administration doses were 8.5 mg/kg body weight for Fe2O3 and 2.5 mg/kg body weight for ZnO nanoparticles. Administering the nanoparticles via a dry powder sprayer coordinated with the respiration of animals has a higher delivery efficiency.
At 12 h and 36 h after the final administration, five rats from each group were sacrificed. Blood samples were taken from an abdominal vein. The brain, olfactory bulb (OB), trachea, lungs, liver, and kidney were collected. Small pieces of each tissue were fixed in 10% formalin for histopathological examination. The remaining tissues were prepared for the determination of Fe and Zn content.

Statistics:

Results were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 11.0 (SPSS Inc., Chicago, USA). A one-way analysis of variance (ANOVA) following the least significant difference (LSD) test was used to compare all groups of animals in the study. A value of P < 0,05 was considered statistically significant.

Sex:

male

Dose descriptor:

other: test dose

Effect level:

8.5 other: mg/kg bw

Remarks on result:

other: after administration of Fe2O3 nanoparticles for 3 days (6 doses of 8.5 mg/kg bw), symptoms of debilitation, anorexia, and coat dullness were observed

Mortality:

No deaths occurred over the entire duration of the experiment.

Clinical signs:

other: After administration of Fe2O3 and ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed in both Fe2O3-treated and ZnO-treated groups.

Animal Health

After administration of Fe2O3 and ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed in both Fe2O3-treated and ZnO-treated groups. However, no deaths occurred over the entire duration of the experiment.

The Content of Fe and Zn in Tissues

The content of Zn and Fe in lung, liver, kidney, brain, and olfactory buib tissues were quantitatively measured using NAA. Fe content in lung and liver tissues were significantly increased in the -treated group after 36 h.

Biochemical Parameters in Serum

The activities of serum ALT, AST, CK, and LDH in both the Fe2O3 -treated group were significantly lower compared to those of the control group (p<0.05) indicated the possibility of liver inflammation from this treatment.

Table 1:  Effects of Fe2O3 and ZnO nanoparticles on serum encyme levels (Mean ± S.D.).
Groups	ALT (U/L)	AST (U/L)	AST/ALT	ALP (U/L)	TP(g/L)	ALB (g/L)	GLβ (g/L)	A/G	CK (U/L)	LDH (U/L)
Control (n = 10)	47 ± 8	123 ± 28	2.6 ± 0.3	649 ± 118	49 ± 1	30 ± 1	19 ± 1	1.6 ± 0 1	1231 ± 348	990 ±383
Fe2O3 - 12 h (n=5)	44 ± 5^a	96 ± 21	2.2 ± 0.3^d	585 ± 60	52 ± 1^ad	32 ± 1^a	20 ± 0.3^ad	1.6 ± 0 1^a	671 ± 92^d	609 ± 299
Fe2O3 - 36 h (n=5)	38 ± 3^d	77 ± 5^d	2.1 ± 0.1^bd	522 ± 63	53 ± 2^bd	32 ± 2^b	21 ± 1^d	1.5 ± 0 1^cd	534 ± 111^d	224 ± 47^d
ZnO - 12 h (n=5)	33 ± 4^de	74 ± 7^d	2.3 ± 0.1	462 ± 92^d	46 ± 2^ad	27 ± 2^ad	19 ± 1^e	1.4 ± 0.1^d	616 ± 146^d	555 ± 221
ZnO - 36 h (n=5)	45 ± 4	80 ± 9^d	1.8 ± 0.04^de	527 ± 49	47 ± 2	27 ± 1^d	20 ± 2	1.4 ± 0.1^d	582 ± 181^d	353 ± 182^d
^ap< 0.05, ZnO post-exposure 12 h versus Fe2O3post-exposure 12h;
^bp< 0.05 ZnO post-exposure 36 h versus Fe2O3post-exposure 36 h,
^cp< 0.05 ZnO post-exposure 12 h versus Fe2O3post-exposure 36 h,
^dp< 0.05 ZnO/Fe2O3-treated groups versus control group.
^ep < 0.05 ZnO/Fe2O3post-exposure 12 h versus 36 h.

Histopathological Evaluation

Exposure to Fe2O3 nanoparticles caused serious hepatic lesions compared to the controls. Inflammation, interstitial hyperemia, fatty degeneration around central vein, and hepatocyte necrosis were noticeable. In general, the pathological changes in liver were more severe in the ZnO-treated group compared to those in Fe2O3 treated group. The pathological lesions of lung tissues show inflammation, interstitial hyperemia, emphysema, and also interstitial substance hyperplasia was evident following exposure to either nanoparticie type. The lung lesions tended to be more severe in the Fe2O3 -treated group. There were no abnormal pathological changes in the trachea, kidney, or brain following administration of either type of nanoparticle.

Table 2 Pathological alterations in rats exposed to Fe2O3and ZnO nanoparticles
Groups	Liver	Kidney	Lung	Trachea
Fe2O3(12 h)
1	+^a	-	++^c	-
2	+^b	-	++^cd	-
3	+^a	-	++^c	-
Fe2O3(36 h)
1	++^f	-	++^cd	-
2	++^b	-	++^c	-
3	+^a	-	++^c	-
ZnO (12 h)
1	++^f	-	+^c	-
2	+^a	-	++^c	-
3	++^e	-	+^c	-
ZnO (36 h)
1	+^a	-	+^c	-
2	++^a	-	+^cd	-
3	++^a	-	+^c	-
Control	-	-	-	-
+ Abnormal
- Normal
^aInflammation and obvious interstitial hyperemia in liver
^bFatty degeneration of hepatocytes around central vein.
^cInflammation, obvious interstitial hyperemia and emphysema in lung.
^dInterstitial substance accrementition in lung
^eSpotty necrosis of hepatocytes
^fLarge-area necrosis of hepatocytes

Executive summary:

The toxic effects of inhalation exposure to ferric oxide (Fe₂O₃) and zinc oxide (ZnO) nanoparticles in rats were investigated. Male Wistar rats were consecutively treated with Fe₂O₃ at 8.5 mg/kg body weight and ZnO nanoparticles at 2.5 mg/kg body weight, twice daily tor 3 days. ZnO and Fe2O3 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer. Content of Fe₂O₃ and ZnO in tissues, biochemical parameters in serum, and histopathological examinations were analyzed at 12 h and 36 h after the 3 day treatment. An extended set of biochemical parameters was measured in serum, which was obtained from an abdominal vein at sacrifice. Blood serum was collected for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), albumin (ALB), and globulin (GLB) by an automalic biochemical analyzer (7170A, Hitachi, Tokyo). The content of Fe and Zn in lung, liver, kidney, olfactory bulb, and brain tissues were determined by neutron activation analysis (NAA). Changes in tissue pathology were also investigated to obtain information on the range of pathological changes that might occur following acute exposure to Fe₂O₃ and ZnO nanoparticles.

After administration of Fe₂O₃ and ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed in both Fe₂O₃-treated and ZnO-treated groups. However, no deaths occurred over the entire duration of the experiment. In the Fe₂O₃-treated group, iron (Fe) content in liver and lung tissues was significantly increased at 36 h. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), and lactate dehydrogenase (LDH) in both nanoparticle-exposed groups were significantly decreased compared to the unexposed controls. Histopathological examination showed lhat both types of nanoparticles caused severe damage in liver and lung tissues. Inflammation, interstitial hyperemia, fatty degeneration around central vein, and hepatocyte necrosis were noticeable in the liver. Inflammation, interstitial hyperemia, emphysema, and interstitial substance hyperplasia were evident in the lung. There were no abnormal pathological changes in the trachea, kidney, or brain. Although this damage progressed in both liver and lung throughout the postexposure period, no significant elevation of serum enzyme activities was observed in response to either nanoparticie type.

Once Fe2O3 nanoparticles entered the body via inhalation, it became systemically available and caused toxic effects in internal organs other than in the lungs. Pulmonary retention, extrapulmonary translocation, and redistribution were considered to be the essential mechanisms of organ damage induced by inhaled nanoparticles. Obvious lesions of liver and lung were induced and the levels of serum ALT, AST, ALP, CK, and LDH were all significantly decreased compared with the control groups.

Reference 3

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Guideline:

other: guideline from the National Institue for Ocuupational Safety and Health

Version / remarks:

This is a guideline itself, intended as a source of information about iron oxide fumes.

Principles of method if other than guideline:

This guideline is intended as a source of information for employees, employers, physicians, industrial hygienists, and other occupational health professionals who may have a need for such information. It does not attempt to present all data; rather, it presents pertinent information and data in summary form.

GLP compliance:

no

Test type:

other: guideline from the National Institue for Ocuupational Safety and Health

Specific details on test material used for the study:

• Formula: Fe2O3
• Synonyms: Ferric oxide fume
• Appearance: Red-brown fume with a metallic taste

Species:

other: not applicable

Route of administration:

inhalation

Sex:

male/female

Dose descriptor:

other: PEL (Permissible Exposure Limit)

Effect level:

10 mg/m³ air

Based on:

test mat.

Exp. duration:

8 h

Remarks on result:

other: current OSHA standard for iron oxide fume

Sex:

male/female

Dose descriptor:

other: Threshold Limit Value

Effect level:

5 mg/m³ air

Based on:

test mat.

Remarks on result:

other: Recommendation of the American Conference of Governmental Industrial Hygienists.

Permissible Exposure Limit (PEL)

The current OSHA standard for iron oxide fume is 10 milligrams of iron oxide fume per cubic meter of air (mg/m³) averaged over an eight-hour work shift The American Conference of Governmental Industrial Hygienists has recommended for iron oxide fume a Threshold Limit Value of 5 mg/m³.

Health Hazard Information

- Routes of exposure: Iron oxide fume can affect the body if it is inhaled.

- Effects of overexposure Repeated exposure to iron oxide fume over a period of years may cause x-ray changes of the lungs, but does not cause the exposed person to become ill.

- Reporting signs and symptoms A physician should be contacted if anyone develops any signs or symptoms and suspects that they are caused by exposure to iron oxide fume.

- Recommended medical surreillance The following medical procedures should be made available to each employee who is exposed to iron oxide fume at potentially hazardous levels:

1. Initial Medical Examination:

- A complete history and physical examination: The purpose is to detect pre-existing conditions that might place the exposed employee at increased risk, and to establish a baseline for future health monitoring. Examination of the respiratory system should be stressed.

- 14** x 17*' chest roentgenogram: Iron oxide fume causes an apparently benign pneumoconiosis. However, since exposure may be associated with other more toxic dust exposures, surveillance of the lungs is indicated.

- FVC and FEV (1 sec): Iron oxide fume causes an apparently benign pneumoconiosis. However, since exposure may be associated with other more toxic dust exposures, periodic surveillance is indicated.

2 Periodic Medical Examination: The aforementioned medical examinations should be repeated on an annual basis, except that an x-ray is considered necessary only when indicated by the results of pulmonary function testing.

Summary of toxicology

Inhalation of iron oxide fume or dust causes an apparently benign pneumoconiosis termed siderosis. Iron oxide alone does not cause fibrosis in the lungs of animals, and the same probably applies to humans. Exposures of 6 to 10 years are usually considered necessary before changes recognizable by x-ray can occur; the retained dust gives x-ray shadows that may be indistinguishable from fibrotic pneumoconiosis. Fight of 25 welders exposed chiefly to iron oxide for an average of 18.7 (range 3 to 32) years had reticulonodular shadows on chest x-rays consistent with siderosis but no reduction in pulmonary function; exposure levels ranged from 0.65 to 47 mg/m8. In another study, 16 welders with an average exposure of 17.1 (range 7 to 30) years also had x-rays suggesting siderosis and spirograms which were normal; however, the static and functional compliance of the lungs was reduced; some of the welders were smokers. The welders with the lowest compliance complained of dyspnea.

Monitoring and Measurement Procedures

• General

Measurements to determine employee exposure are best taken so that the average eight-hour exposure is based on a single eight-hour sample or on two four-hour samples. Several short-time interval samples (up to 30 minutes) may also be used to determine the average exposure level. Air samples should be taken in the employee's breathing zone (air that would most nearly represent that inhaled by the employee).

• Method

Sampling and analyses may be performed by collection of iron oxide fume on a filter, followed by atomic absorption spectrophotometric analysis. An analytical method for iron oxide fume is in the NIOSH Manual of Analytical Methods, 2nd Ed., Vol. 4, 1978.

Respirators

• Good industrial hygiene practices recommend that engineering controls be used to reduce environmental concentrations to the permissible exposure level. However, there are some exceptions where respirators may be used to control exposure. Respirators may be used when engineering and work practice controls are not technically feasible, when such controls are in the process of being installed, or when they fail and need to be supplemented. Respirators may also be used for operations which require entry into tanks or closed vessels, and in emergency situations. If the use of respirators is necessary, the only respirators permitted are those that have been approved by the Mine Safety and Health Administration (formerly Mining Enforcement and Safety Administration) or by the National Institute for Occupational Safety and Health.

• In addition to respirator selection, a complete respiratory protection program should be instituted which includes regular training, maintenance, inspection, cleaning, and evaluation.

Common Operations And Controls

The following list includes some common operations in which exposure to iron oxide fume may occur and control methods which may be effective in each case:

Operation:

Liberation in production of steel ingots;

processing of iron ore to pig iron;

heating and pouring of molten metal in foundry operations;

hot rolling sheet and strip steel;

fettling of castings in foundry operations;

during forging of metal items containing iron/steel;

pressing of metal items, grinding and polishing of glass, precious metals, stones, and gem stones

Controls:

local exhaust ventilation; general dilution ventilation; personal protective equipment

Emergency First Aid procedures

In the event of an emergency, institute first aid procedures and send for first aid or medical assistance.

• Breathing

If a person breathes in large amounts of iron oxide fume, move the exposed person to fresh air at once. If breathing has stopped, perform artificial respiration. Keep the affected person warm and at rest. Get medical attention as soon as possible.

• Rescue

Move the affected person from the hazardous exposure. If the exposed person has been overcome, notify someone else and put into effect the established emergency rescue procedures. Do not become a casualty. Understand the facility's emergency rescue procedures and know the locations of rescue equipment before the need arises.

Spill Procedures

• Persons not wearing protective equipment and clothing should be restricted from areas of releases until I cleanup has been completed.

• If potentially hazardous amounts of iron oxide fume are inadvertantly released, ventilate the area of the release to disperse the fume.

RESPIRATORY PROTECTION FOR IRON OXIDE FUME
Condition	Minimum Respiratory Protection^aRequired Above 10 mg/m³
Particulate Concentration
100 mg/m³ or less	Any fume respirator or high efficiency particulate filter respirator.
	Any supplied-air respirator.
	Any self-contained breathing apparatus.
500 mg/m³ or less	A high efficiency particulate filter respirator with a full facepiece.
	Any supplied-air respirator with a full facepiece, helmet or hood.
	Any self-contained breathing apparatus with a full facepiece.
5,000 mg/m³ or less	A powered air-purifying respirator with a high efficiency particulate filter.
	A Type C supplied-air respirator operated in pressure-demand or other positive pressure or continuous-flow mode.
Greater than 5,000 mg/m^3or entry and escape from unknown concentrations	Self-contained breathing apparatus with a full facepiece operated in pressure-demand or other positive pressure mode.
	A combination respirator which includes a Type C supplied-air respirator with a full facepiece operated in pressure-demand or other positive pressure or continuous-flow mode and an auxiliary self-contained breathing apparatus operated in pressure-demand or other positive pressure mode.
Fire Fighting	Self-contained breathing apparatus with a full facepiece operated in pressure-demand or other positive pressure mode.
*Only NIOSH-approved or MSHA-approved equipment should be used.

Conclusions:

The Permissible Exposure Limit (PEL) for iron oxide fume is 10 mg/m3 air (8 h exposure) (OSHA standard)
The Threshold Limit Value of iron oxide fume is 5 mg/m3 air (Recommendation of the American Conference of Governmental Industrial Hygienists)

Executive summary:

The Permissible Exposure Limit (PEL) for iron oxide fume is 10 mg/m3 air (8 h exposure) (OSHA standard)

The Threshold Limit Value of iron oxide fume is 5 mg/m3 air (Recommendation of the American Conference of Governmental Industrial Hygienists)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity other routes:

The acute toxicity of iron gluconate complexes was evaluated in several experiments with injection type administrations. Although these administration routes are not relevant for the REACH registration of the registered substance, the below results are supportive to elucidate the acute toxicity of iron glucoheptonate.

Data on sodium ferric gluconate complex in sucrose injection

The principal read across substance ferrous gluconate is also available as FERRLECIT (sodium ferric gluconate complex in sucrose injection), which is a stable macromolecular complex used to replete and maintain the total body content of iron. Acute toxicity studies available for Ferrlecit had been carried out in mice, rats, rabbits and dogs. Ferrlecit® at elemental iron doses of 125 mg/kg, 78.8 mg/kg, 62.5 mg/kg and 250 mg/kg caused deaths to mice, rats, rabbits, and dogs respectively. The major symptoms of acute toxicity were decreased activity, staggering, ataxis, inceases in the respiratory rate, tremor, and convultions.

The LC50 value formale micewas159 mg Fe/kg, the one forfemale micewas155 mg Fe/kg.

After administration of 62.5 - 157.5 mg Fe/kg, the LC₅₀value formaleratswas111.25 mg Fe/kg, the one forfemale ratswas90 mg Fe/kg. After 5 days: combined male/female274 mg Fe/kg.

After administration of doses of 62.5. 87.5 and 112.5 mg Fe/kg, the LC₅₀value forrabbitsafter 5 days was70.4 mg Fe/kg.

After administration of doses of 125 - 250 mg Fe/kg, the LC₅₀value for dogs was262.5 mg Fe/kgafter 24 hours. After administration of 5 mL (= 62.5 mg Fe/kg) no systemic toxicity was found in dogs after 96 hours.

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Data on ferrous gluconate

This report is concerned with the animal toxicities of a new high molecular iron-carbohydrate complex compared to those of several other preparations (Weaver, 1961). For this purpose several toxicity test were conducted in different animal species. In the acute toxicity test in mice and dogs the compounds (i.e. ferrous gluconate) were administered as aqueous solutions where possible, otherwise as fine suspensions. Groups of 10 or more male albino Swiss-Webster mice were given the compounds intravenously (i.v.), intraperitoneally (i.p.), or intragastrically (i.g.). The rate of i.v. injections was 0.01 mL per second. in addition, mongrel dogs, unselected as to sex, were used in all acute toxicity studies. Acute, rapid, i.v. injections were made and the LD₅₀determined at 24 hours.

The animals were observed closely for several hours following injection, and the LD₅₀and 95% confidence limits were determined at the end of 24 hours by the method of Litchfield and Wilcoxon. The animals receiving iron-carbohydrate complex and ferrous sulfate were observed for a period of 7 days following injection and any delayed manifestations of toxicity were recorded. If any deaths occurred after 24 hours, the LD₅₀was recalculated at the end of the 7-day observation period. Strain and sex differences in response to iron-carbohydrate complex and ferrous sulfate were evaluated by the use of female albino Swiss-Webster, black female BDF-1 and C-57 and female fawn DBA-2 mice.

Since the iron content of the compounds varies considerably comparisons were made on the basis of actual iron content. The iron polysaccharide complex was the least toxic by the i.v. route in mice and iron-carbohydrate complex was next. The other compounds tested were 2 to 4 times as toxic as the iron-carbohydrate complex. There was no significant difference between the 1- and 7-day toxicities for either iron-carbohydrate complex or ferrous sulfate for the i.v. and i. g. routes in mice. In Swiss-Webster male mice thei.v. LD₅₀was 199 mg/kgfor ferrous gluconate. Iron-carbohydrate complex showed a low order of toxicity in 3 species of laboratory animals.

The iron-carbohydrate complex was the least toxic of the compounds studied i.p. in mice, though not significantly less toxic than the iron polysaccharide complex. It had only 1/10 the toxicity of ferrous sulfate. In Swiss-Webster male mice thei.p. LD₅₀was 160 mg/kgfor ferrous gluconate.

Following i.v. administration in dogs, ferric choline citrate and ferrous sulfate were the most toxic of the compounds tested. Ferrous gluconate and iron-carbohydrate complex were the least toxic. The iron polysaccharide complex was given in a dose of 40 mg/kg to 3 dogs without lethal effects. Because of the fixed concentration of the solution, the volume necessary for higher dosages was too great to be practical.

Emetic responses to the various iron compounds in dogs showed iron-carbohydrate complex to be less gastrointestinal distress as indicated by emesis than any of the other compounds. Ferrous sulfate and ferroglycine sulfate complex were the most emetic in this study.

Thus, iron-carbohydrate complex showed a low order of toxicity in 3 species of laboratory animals. Studies in mice indicate that iron-carbohydrate complex is less toxic than ferrous sulfate, ferrous gluconate, ferrous fumarate, ferroglycine sulfate complex, ferric choline citrate and iron polysaccharide complex by the oral and intraperitoneal routes.

Gastric intolerance as indicated by emesis was very much less for iron-carbohydrate complex than for the other 5 compounds. in summary, none of the compounds tested was less toxic than iron-carbohydrate complex by the intravenous route in dogs and iron-carbohydrate complex produced the least gastrointestinal irritation as indicated by emesis in the dog.

The acute toxicity of ferrous gluconate was determined in direct comparison with that of ferrous sulfate following both intravenous and oral administration in male, albino Swiss mice weighing 22 ± 2 g (Hoppe, 1955). For the intravenous injection, the compounds were administered in aqueous solution in a volume of 0.01 cc./g bw at a rate of 1.0 cc./minute. All animals were housed in air-conditioned quarters with food and water available at all times, with the exception of the period immediately preceding the oral medications. The mice were fasted for 4 hours before oral administration of the ferrous gluconate and ferrous sulfate dosages. The mice were observed closely for several hours following injection, and the LD₅₀± SE was estimated at the end of 24 hours by the method of Miller and Tainter. The animals were held under close observation for a period of one week following injection and any delayed manifestations of toxicity were recorded. Where delayed deaths occurred after 24 hours, the LD₅₀was recalculated at the end of the 7-day observation period.

Intravenous administered ferrous sulfate was found to be approximately twice as toxic in mice as ferrous gluconate in terms of the salt (LD₅₀values of 65 ± 4.8 versus114 ± 7.6 mg/kg after 24 hoursand 51and 98 mg/kg after 7 days, respectively). When the data were calculated in terms of ferrous iron, there did not appear to be any apparent difference in the acute intravenous toxicity of these two compounds in mice. The value of 13 ± 1 mg/kg for ferrous sulfate is in almost precise agreement with the value, 13.8 mg/kg of iron, reported for ferrous sulfate in mice by Edge and Somers. Although a majority of the mice died in the first 24 hours following injection, several deaths occurred in the next 3 to 5 days. No deaths occurred after 5 days. The LD₅₀value for ferrous gluconate at 7 days was not significantly different from the 24-hour value. The 7-day LD₅₀value for ferrous sulfate, however, indicated a significant increase in toxicity due to delayed deaths. In the acute deaths, the mice were severely depressed and lapsed into complete prostration which terminated in a brief clonic convulsive episode with cessation of respiration preceding cardiac arrest. A majority of the acute deaths occurred in one to five minutes after intravenous injection.

It is noteworthy, that the acute toxicity of ferrous gluconate is almost twenty times larger, when administered not via intravenous injection but via oral administration (LD₅₀oral for ferrous gluconate, 3700 ± 145 mg/ kg versus the above mentioned LD₅₀of 114 ± 7.6 mg/kg after 24 hours)

The magnitude of the acute oral toxicity values when compared with the acute intravenous figures in mice indicates a relatively low order of absorption from the intestinal tract. An additional safety factor is evident from the oral studies in the cat and the dog in which the local irritant effects induce a protective emesis. These data suggest prompt, gentle gastric lavage along with supportive therapy for shock as an effective emergency measure in those cases where, for any reason, vomiting does not occur spontaneously following oral ingestion of ferrous sulfate, ferrous gluconate or other soluble iron salts.

Justification for classification or non-classification

Based on the results of the available studies, the registered substance does not meet criteria for classification and labelling in accordance with European Regulation (EC) No 1272/2008.