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Basic toxicokinetics

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Endpoint:
basic toxicokinetics
Type of information:
other: EU RAR Risk assessment
Adequacy of study:
weight of evidence
Study period:
2009
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Risk assessment on another substance that provides background to the dissociation or degradation products in water of KMPS. When KMPS triple salt is dissolved in water, it will dissociate and will eventually form K+, SO42- and H2O2, respectively. The dissociation products K+ and SO42- are chemically and biologically not further degradable because they constitute simple basic structures of inorganic nature, which cannot be broken down any further. Furthermore, both ions are physiologically essential elements of all living organisms. KMPS triple salt will form hydrogen peroxide on degradation which is considered to be the active principle of KMPS triple salt and which is considered to be the substance of toxicological relevance in this case.In view of the degradation and dissociation of KMPS triple salt into K+ and SO42- ions as well as to H2O2, it is considered justified, to focus on these entities only for the evaluation of the toxicokinetics and metabolic behaviour of KMPS triple salt.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
other: EU RAR Risk assessment
Title:
Hydrogen Peroxide
Year:
2009

Materials and methods

Objective of study:
not specified
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Expert statement
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentapotassium bis(peroxymonosulphate) bis(sulphate)
EC Number:
274-778-7
EC Name:
Pentapotassium bis(peroxymonosulphate) bis(sulphate)
Cas Number:
70693-62-8
Molecular formula:
H3K5O18S4
IUPAC Name:
pentapotassium bis(peroxymonosulphate) bis(sulphate)
Radiolabelling:
no

Test animals

Species:
other: not applicable; no animal testing
Strain:
other: not applicable; no animal testing
Sex:
not specified
Details on test animals or test system and environmental conditions:
No test animals, expert statement

Administration / exposure

Route of administration:
other: not applicable; no animal testing
Vehicle:
other: not applicable; no animal testing
Details on exposure:
No animal testing, expert statement
Duration and frequency of treatment / exposure:
No animal testing, expert statement
Doses / concentrations
Remarks:
Doses / Concentrations:
No animal testing, expert statement
No. of animals per sex per dose / concentration:
No animal testing, expert statement
Control animals:
other: No test animals, expert statement
Positive control reference chemical:
No animal testing, expert statement
Details on study design:
No animal testing, expert statement
Details on dosing and sampling:
No animal testing, expert statement
Statistics:
No animal testing, expert statement

Results and discussion

Preliminary studies:
No animal testing performed.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
When KMPS triple salt is dissolved in water, it will dissociate and will eventually form K+, SO42- and H2O2, respectively. The dissociation products K+ and SO42- are chemically and biologically not further degradable because they constitute simple basic structures of inorganic nature, which cannot be broken down any further. Furthermore, both ions are physiologically essential elements of all living organisms. KMPS triple salt will form hydrogen peroxide on degradation which is considered to be the active principle of KMPS triple salt and which is considered to be the substance of toxicological relevance in this case.In view of the degradation and dissociation of KMPS triple salt into K+ and SO42- ions as well as to H2O2, it is considered justified, to focus on these entities only for the evaluation of the toxicokinetics and metabolic behaviour of KMPS triple salt.
Details on distribution in tissues:
In view of the degradation and dissociation of KMPS triple salt into K+ and SO42- ions as well as to H2O2, it is considered justified, to focus on these entities only for the evaluation of the toxicokinetics and metabolic behaviour of KMPS triple salt.
Details on excretion:
In view of the degradation and dissociation of KMPS triple salt into K+ and SO42- ions as well as to H2O2, it is considered justified, to focus on these entities only for the evaluation of the toxicokinetics and metabolic behaviour of KMPS triple salt.

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
When KMPS triple salt is dissolved in water, it will dissociate and will eventually form K+, SO42- and H2O2, respectively. The dissociation products K+ and SO42- are chemically and biologically not further degradable because they constitute simple basic structures of inorganic nature, which cannot be broken down any further. Furthermore, both ions are physiologically essential elements of all living organisms. KMPS triple salt will form hydrogen peroxide on degradation which is considered to be the active principle of KMPS triple salt and which is considered to be the substance of toxicological relevance in this case.In view of the degradation and dissociation of KMPS triple salt into K+ and SO42- ions as well as to H2O2, it is considered justified, to focus on these entities only for the evaluation of the toxicokinetics and metabolic behaviour of KMPS triple salt.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: Based on the toxicokinetic evaluation of the decay products of KMPS triple salt, no bioaccumulation is expected.
The chemical nature of KMPS triple salt is characterised by oxidation at the site of first contact. As a consequence thereof, KMPS triple salt will rapidly degrade and will eventually form potassium and sulfate ions and hydrogen peroxide which are the relevant chemical entities to be considered for the assessment of the toxicokinetics and metabolism of KMPS triple salt.
Based on these fundamental properties of KMPS triple salt, it wll be not bioavaiable, neither by inhalation, ingestion, or contact by skin. Bioaccumulation is unlikely.
Executive summary:

 Toxicokinetic Analysis of KMPS triple salt

 

KMPS triple salt is an inorganic salt with a molecular weight of 614.76. It decomposes on heating without a definite melting point. A boiling point is also not available, as KMPS triple salt decomposes at temperatures above 70 °C. KMPS triple salt is freely water soluble: 360 g/L at 20 °C. The vapour pressure was determined to be < 1.7x10E-4 Pa at 25 °C. The partition coefficient of KMPS triple salt was determined to a log Pow of < 0.3 at 20 °C. Due to its low vapour pressure and its properties as inorganic salt, an exposure by inhalation is not very likely. As showed by the repeated dose inhalation toxicity study, no systemic effects have occurred. Absorption by the skin is also not very likely, as no systemic effects have been detected in the skin toxicity studies, showing the major action as local effects due to the rapid decomposition of KMPS triple salt by formation of hydrogen peroxide, which is the main operation principle. Therefore, examination of the toxicokinetic action of the decay products of KMPS triple salt is the basis for the examination of the toxicokinetic properties of KMPS triple salt.

Hydrogen Peroxide

Besides potassium and sulfate, KMPS triple salt will additionally form hydrogen peroxide on degradation which is considered to be the substance of toxicological relevance in this case. In addition, hydrogen peroxide which is known to be a strong oxidant, is further considered to be the active principle in KMPS triple salt which is supported by the “Opinion of the Scientific Committee on Consumer Products on Hydrogen peroxide, in its free form or when released, in oral hygienc products or tooth whitening products” in which KMPS triple salt was identified as a hydrogen peroxide releaser and in which it was stated that KMPS triple salt should be regulated similarly as hydrogen peroxide on the basis of hydrogen peroxide or reactive oxygen products released. Due to the high reactivity of KMPS triple salt and as KMPS triple salt and hydrogen peroxide reveal the same mechanism of action, i.e. oxidation and irritation/corrosion at the site of first contact, it is considered justified to use the available information on the toxicokinetics and metabolism of hydrogen peroxide from the EU Risk Assessment Report on hydrogen peroxide for an assessment of the ADME of KMPS triple salt. The applicant is, therefore, of the opinion, that the available ADME data on hydrogen peroxide also satisfactorily address the fate of KMPS triple salt and further studies on the toxicokinetics and metabolism of KMPS triple salt are, thus, not required.

Publicly available information on the well-characterised toxicokinetics and metabolism of hydrogen peroxide which further substantiates that no kinetic and metabolism studies with KMPS triple salt are required has been excerpted from the EU RAR of hydrogen peroxide. These data from the EU RAR are considered to satisfy this requirement and details on the toxicokinetics and metabolism of hydrogen peroxide are described below:

Endogenous Occurrence:

Hydrogen peroxide is a normal metabolite in aerobic cells. It has been stated that the cellular concentration is regulated at 10-9-10-7M depending on the balance between formation and degradation. In normoxia, the rate of H2O2production in the liver of normal anesthetised rats was measured as 380 nmol H2O2/min per g of liver. The total estimated production of 1,450 nmol/min per 100 g rat would indicate that about 75% of all H2O2generated may be attributed to the liver. The effective rate of H2O2formation depends on the substrate and oxygen supply. Production is markedly enhanced in the hyperbaric environment. Studies with isolated fractions of rat liver suggest that mitochondria (through a variety of enzymic reactions leading to univalent or divalent reduction of oxygen), microsomes (through normal electron transport reactions, glycolate oxidase, D-aminoacid oxidase, urate oxidase), peroxisomes (throughβ-oxidation of fatty acids), and soluble enzymes provide 14%, 47%, 34% and 5%, respectively, of the cytosolic H2O2when fully supplemented with their substrates.

Apart from intermediary metabolism, hydrogen peroxide together with other reactive oxygen species plays a role in cellular defences against invading organisms. H2O2generated by phagocytes can destroy normal or malignant cells and alter erythrocyte, platelet, neutrophil or lymphocyte function. As an oxidant hydrogen peroxide is unusual because (1) it reacts slowly with organic substrates and thus can diffuse at a certain distance in biological systems, (2) its small size and lack of charge facilitate its movement across plasma membranes, and (3) its intracellular concentration is controlled by several enzymes. As the plasma membrane of the polymorphonuclear neutrophil encircles an opsonised particle, superoxide anion and hydrogen peroxide are released directly into the extracellular medium until the particle is completely engulfed. Inside this environment hydrogen peroxide may remain in the vacuole or diffuse either into the cytoplasm or outside the cell. H2O2equilibrium will be regulated by the relative amounts of substance generated or consumed in each of these sites, transiently the concentrations may reach 10μM.

There is uncertainty about the true levels of hydrogen peroxide in biological media due to analytical difficulties. Investigation of published spectrophotometric and HPLC techniques for analysing hydrogen peroxide in human and dog plasma showed that the measured substance probably was not H2O2but most likely peroxides because (1) catalase treatment did not abolish the peaks (in contrast to H2O2control), and (2) added exogenous hydrogen peroxide did not markedly increase the peak area. Blood and plasma of humans and rats were analysed for hydrogen peroxide with a radioisotopic method. Among six male laboratory workers, aged 30-35 years, H2O2concentrations in the whole blood ranged from 114 to 577μM (reflecting the high levels in phagocytic cells) and in the plasma from 13 to 57μM. The corresponding H2O2concentrations in rat blood samples were similar. Data on hydrogen peroxide concentrations in human exhaled air have given values ranging from non-detectable or maximally 0.5x10-8 M to 0.5μM; the methods of sampling and analysis have varied. A normal human subject breathing normally had 1–3x10-8M H2O2in breath (measured as chemiluminescence intensity). The breath luminescence increased greatly after breathing pure oxygen and 5 minutes after smoking a cigarette in the morning. It was presumed that smoking activated macrophages in the lung, releasing H2O2. The human aqueous humour is reported to contain normally 19-31μM of H2O2, and similar concentrations have been measured in the corresponding primate and bovine samples. Among 17 cataract patients hydrogen peroxide levels in aqueous humour ranged from 10 to 660μM.

Obviously the biological functions for hydrogen peroxide and other reactive oxygen species require strict regulation of concentration in various intracellular and body compartments (see below).

Absorption and Distribution:

Biological membranes are highly permeable to H2O2; the permeability constants of 0.2 cm/min for peroxisomal membranes and 0.04 cm/min for erythrocyte plasma membranes may be compared with those for water in a variety of membranes, ranging from 0.02 to 0.42 cm/min. Thus, hydrogen peroxide is expected to be readily taken up by the cells constituting the absorption surfaces, but at the same time it is effectively metabolised, and it is uncertain to what extent the unchanged substance may enter the blood circulation. Moreover, in the blood the red blood cells have an immense metabolic capacity to degrade hydrogen peroxide.

Absorption from mucous membranes:

Administration of hydrogen peroxide solutions to body cavities lined by mucous membranes, such as sublingually, intraperitoneally and rectally resulted in increased oxygen content of the draining venous blood and, if the amounts of hydrogen peroxide were sufficiently high, formation of oxygen bubbles. Mongrel dogs were treated with colonic lavage, or the lavage of small and large bowel was performed through an enterotomy with dilute saline solutions of hydrogen peroxide. Small amounts of the more concentrated solution (1.5% or higher) produced immediate whitening of the mucosa, with prompt appearance of bubbles in the circulation. More dilute (0.75-1.25%) solutions had the same effect when left in contact with the bowel for a longer time or when introduced under greater pressure or in greater volume for a given length of bowel. Venous bubbling was never observed at concentrations less than 0.75% H2O2. In none of the animals did mesenteric thrombosis or intestinal gangrene develop. Application of 1% hydrogen peroxide to the serosal membrane caused whitening due to gas filled small vessels; higher concentrations (up to 30%) on the skin and mucous membranes (of various species) caused lasting damage when subcutaneous emphysema and disturbances of local blood circulation impaired tissue nutrition. In two cats, sublingual application of 1.5 ml of 9%18O-labeled hydrogen peroxide or 0.1 ml 19%18O-labeled hydrogen peroxide was followed up with mass spectrometric analyses in arterial (femoral artery) blood and exhaled air. Within about one hour in the former case, and within half an hour in the latter case, 1/3 of the labeled oxygen administered was exhaled. There was a rapid initial rise of the arterial blood18O-concentration, but the arterial blood oxygen saturation gradually declined, probably because of impaired gas exchange in the lung due to oxygen embolism.

Absorption from the lungs:

Anesthetised rabbits were administered 1-6% hydrogen peroxide aerosol by inhalation. The left atrial blood was found to be supersaturated with oxygen up to levels that corresponded to oxygen administration at 3 atm. When this level was increased, small bubbles began to appear in the samples. The 1% aerosol, which was least irritating, provided as high arterial oxygen levels as the higher hydrogen peroxide concentrations. Concerning acute inhalation toxicity studies, it is not clear whether the mechanisms of lethal effect are local or systemic. However,) in a poorly documented study it was stated that the LC50level for rats derived from 4-hour exposure to hydrogen peroxide vapours was 2,000 mg/m³ (inhalation and whole-body shaved skin exposure), and that the primary cause of death was gas (oxygen) embolism.

Conclusions from experimental studies:

Animal studies show that administration of high concentrations of hydrogen peroxide by various routes, resulting in high rates of absorption, leads to oxygen bubble formation in blood vessels. This indicates indirectly that hydrogen peroxide has been absorbed systemically but then rapidly degraded by metabolising enzymes in the circulating blood. Under some conditions systemic embolisation of oxygen (micro)bubbles has been found to occur. This was demonstrated in a study of a new technique for oxygenating blood in which 3% H2O2in normal saline was infused at a controlled rate into the right ventricle of 12 pigs whose blood catalase activity was only slightly less than in humans. The rate of infusion was limited by bubble formation leading to pulmonary and systemic embolisation. In a further study performed it was demonstrated that the intravenous acute toxicity of hydrogen peroxide in rabbits was inversely related to the substance concentration (the studied range of dilutions was 3.6-90%). With successive dilutions the blocking effects at the injection site were less, allowing hydrogen peroxide-derived oxygen bubbles to be distributed in the blood circulation and to cause more toxicity, as evidenced by convulsions, and more deaths.

Observations from human incidents related to absorption and distribution:

There are two reported cases of accidental ingestion of 35% hydrogen peroxide which resulted in brain injury presumed to be due to cerebral oxygen embolism. The latter of these cases was more convincing as it concerned a specific pattern of multiple cerebral infarctions (detected with MRI) occurring immediately after the ingestion. The authors speculated on the pathophysiologic mechanism: a patent foramen ovale of the heart (not said to the be involved in the case), some unmetabolised hydrogen peroxide crossing the pulmonary capillary bed into the arterial circulation, or aspiration and absorption of hydrogen peroxide from the pulmonary capillaries. In a third case a child ingested about 230 g of 3% hydrogen peroxide solution. He was found dead 10 hours later and gas emboli were found in the intestinal lymphatics and the pulmonary vasculature. Moreover, there were clear vacuoles in the spleen, kidney and myocardium.

Hydrogen peroxide has often been used for irrigation of surgical wounds. A 54-year-old male received irrigation under pressure of an infected and fistulous herniorrhaphy wound with 5-20 ml volume of 3% hydrogen peroxide. Not all irrigating volume seemed to have drained from the wound. On the fifth irrigation the patient suddenly lost consciousness, showed cardiac shock and fell to coma which lasted 15 minutes. There was no indication of red cell damage. ECG showed signs of transient myocardial ischaemia. The patient made a full recovery within 3 days. The authors proposed that the most likely mechanism of this occurrence was widespread embolisation of oxygen microbubbles, especially to the cerebral and coronary arteries. Two patients had their right thoracic cavity irrigated with 300 ml of 3% hydrogen peroxide during lung surgery. After one of the patients had showed clinical signs of pulmonary embolism, the other patient was monitored with transoesophageal echocardiography. Within some seconds after the irrigation bubbles were detected in the right atrium and ventricle lasting for about 3 minutes. The patient did not show any haemodynamic or respiratory complications, however. The authors cited four further case reports of gas embolism (involving five patients) in the context of surgical irrigation of body cavities with hydrogen peroxide, and two further case reports were subsequently located. Thus, hydrogen peroxide may be particularly dangerous in surgical operations when used in closed spaces or under pressure, where liberated oxygen cannot escape.

Metabolism:

Detoxification (scavenging) reactions:

There are two main hydrogen peroxide metabolising enzymes, catalase and glutathione peroxidase, which control H2O2concentration at different levels and in different parts of the cell. Catalase deals with large amounts of H2O2that may be generated in peroxisomes. Glutathione peroxidase (GSH peroxidase) metabolises H2O2in both the cytosolic and mitochondrial compartments. A variety of small molecule, nonenzymatic antioxidants complete an efficient intra- and extracellular network of defences such as vitamin E, ubiquinols, carotenoids, ascorbic acid and glutathione.α-Keto acids such as pyruvate nonenzymatically reduce hydrogen peroxide to water while undergoing decarboxylation at the 1-carbon position and may thus function as efficient scavengers of H2O2. The kinetics of removal for extracellular H2O2was examined in cultured fibroblasts. The process involved two kinetically different reactions, the first one being characterised by a relatively low Km value (about 40μM), the second one showed a linear dependence of the rate up to 500μM. By using specific inhibitors, it could be concluded that the first reaction involved GSH peroxidase and the second catalase. It was inferred that 80-90% of H2O2is decomposed by GSH peroxidase at hydrogen peroxide levels lower than 10μM. The contribution of catalase increases with the increase of H2O2concentration.

The activities of catalase and GSH peroxidase are unevenly distributed in various tissues and across different species. The brain, lung and heart have low catalase activities while the muscle tissue is lacking effective concentration of GSH peroxidase. Measurement of antioxidant enzyme activities in the rat gastrointestinal tract showed that the specific activity of glutathione peroxidase was maximal in the stomach while catalase activity was uniform in all regions of the g-i tract. There was no change in activity by age. The maximum activities were located in the cell cytosol. Various antioxidant enzyme activities were measured in lung homogenates from rats, hamsters, baboons and humans. Glutathione peroxidase was higher in rat lung than in baboon or hamster lung. Catalase activity was variable, being 10 times higher in baboons than in rats. Rat lung antioxidant enzyme activities were different from the other species. Hamster seemed to mimic most closely humans.

Studies in freshly isolated human bronchial epithelial cells indicated significant antioxidative capacity. Inactivation of both catalase and glutathione reductase (resulting in impaired glutathione redox cycle) made the cells more susceptible to hydrogen peroxide-mediated injury. However, in another study, most human volunteers who were exposed for about 15 hours to 100% oxygen developed tracheobronchitis due to oxidant toxicity but the genes for the major antioxidant enzymes (superoxide dismutases and catalase) were expressed at very low levels and were not upregulated by exposure. Catalase activity in the human bronchial epithelium at baseline was 0.008±0.002 U/10E+6 cells and did not change significantly after exposure to 100% oxygen.

Selective inhibition of catalase and glutathione reductase activities in freshly isolated and cultured human alveolar macrophages demonstrated that catalase was the bulk hydrogen peroxide scavenger; however the glutathione redox cycle was more important in maintaining cell membrane integrity. The primary localisation of catalase was in peroxisomes, and there were low levels in the cytoplasmic and nuclear matrices. Even a highly efficient catalase activity in the cell membrane was speculated but not proven.

Freshly isolated rabbit lung alveolar type II pneumocytes (ATII) were coincubated with either hydrogen peroxide generating xanthine-xanthine oxidase system yielding about 300μM H2O2at steady-state, or with 300μM H2O2for up to one hour. Cellular metabolic defences were modified either by inhibition of catalase with aminotriazole or by conjugation of reduced glutathione with chlorodinitrobenzene. ATII cells cleared H2O2at a higher rate than an equivalent amount of free catalase. Aminotriazole decreased ATII cell catalase activity by 89% and prolonged the clearance half-life of H2O2from 1.3 min to 18.1 min; the treated cells were more susceptible to oxidant injury, as shown by their decreased ability to exclude trypan blue after 60 min of H2O2exposure. Glutathione-depleted cells scavenged H2O2at the same rate as controls. Hence ATII cells reduce the extracellular hydrogen peroxide (at high physiological concentrations) mainly by a catalase-dependent pathway. ATII cells secrete surfactant and actively transport sodium across the alveolar space. They are a minor component of the alveolar epithelial surface that is mainly (>95%) made up of alveolar type I cells. While ATII cells are resistant to exogenous oxidants, ATI cells are more sensitive. In conclusion, ATII cells play an important role in protecting the alveolar epithelium from injury by high H2O2concentrations via a predominantly catalase-dependent process.

In the blood red blood cells efficiently remove intracellular and extracellular hydrogen peroxide. Under physiologic conditions the ability of the red blood cells to protect haemoglobin from oxidation depends largely on the presence or absence of glucose and its utilisation for the production of NAD(P)H and maintenance of sufficient levels of reduced glutathione. Glutathione peroxidase is of major importance in this scheme. If additional (exogenous) sources of peroxide formation are present, catalase concentration in the red cell becomes important. Under these conditions formation of methaemoglobin by peroxide depends on catalase concentration: the higher the catalase activity, the more resistant the cell. Another study demonstrated that human red blood cells efficiently removed extracellular hydrogen peroxide and protected the surrounding tissue against damage mediated by peroxide and its secondary products hydroxyl radical and hypochlorous acid. The scavenger capacity depended on catalase wheras haemoglobin, GSH and glucose metabolism contributed only minimally. The red cells were approximately one quarter as efficient at removing H2O2as an equivalent concentration of free catalase, i.e. the potential of red cells to remove hydrogen peroxide from blood is immense. Catalase activity in human erythrocytes is 3,600-fold higher than in serum. Serum catalase was somewhat lowered (0.62) in patients of nonhaemolytic anaemia and increased in patients of haemolytic anaemias (8.3-fold) and in pernicious anaemia (6.6-fold).

The brain has low concentrations of catalase and glutathione peroxidase. Dopaminergic neurons of the striatum are exposed to relatively high concentrations of reactive oxygen species, including hydrogen peroxide, during the metabolism of dopamine. The vulnerability of neurons to hydrogen peroxide was found to be attenuated by the presence of glial cells. Exposure of striatal neurons (from mouse embryos) for 30 min to hydrogen peroxide led to a concentration-dependent (10-1,000μM) decrease of cell viability. Toxic effect of 100μM was totally prevented by added catalase or glutathione peroxidase in the presence of reduced glutathione. The capacity of striatal neurons to remove external H2O2(100μM) was 46±6 nmol/mg protein/min. Differential inhibition of catalase or glutathione peroxidase (decreased content of reduced glutathione) indicated that the neuronal defence was mediated primarily by glutathione peroxidase. The viability of striatal astrocytes was not affected by exposure to hydrogen peroxide (up to 1 mM for 60 min), and the neurotoxic effect on the neuronal population was markedly decreased in astrocytoneuronal cocultures. A significant neuroprotection was detectable for 1 astrocyte to about 20 neurons. The capacity of striatal astrocytes to remove external H2O2(100μM) was 317±27 nmol/mg protein/min, i.e. sevenfold higher than the corresponding capacity of neurons. Most of this hydrogen peroxidase activity was attributable to catalase. The protective role of astrocytes was due to its high clearance capacity of hydrogen peroxide rather than a possible release of protective compounds. Since the simultaneous inhibition of both hydrogen peroxidase activities did not completely suppress the clearance of H2O2in either cell type, a nonenzymatic process, such as the Fenton reaction, could also contribute to the disappearance of hydrogen peroxide.

Metabolism related to toxicity:

In the aerobic cellular metabolism complete reduction of a molecule of oxygen to water requires four electrons, and in a sequential univalent process the superoxide anion radical (O2-), hydrogen peroxide and the hydroxyl radical (OH•) intermediates are formed. In the presence of reduced metal ions (Fe2+; Cu+) hydroxyl radicals may originate from hydrogen peroxide by the Fenton reaction. The chemical reactions involved in the generation of reactive oxygen species are shown below.

Molecular oxygen is reduced to water by four one-electron reduction steps:

O2+ e → O2•                                    (superoxide anion)

O2• + e + 2H+→ H2O2                     (hydrogen peroxide)

H2O2+ e + H+→ OH• + H2O             (hydroxyl radical)

OH• + e + H+→ H2O                         (water)

Net: O2+ 4 e + 4 H+→ 2 H2O

Several enzymes are involved in the elimination of (reactive) oxygen species:

SOD

2 O2• + 2H+→ H2O2+ O2               (SOD:superoxide dismutase)

GSPx

H2O2+ 2 GSH → 2 H2O + GSSG    (GSPx:glutathione peroxidase)

CAT

2 H2O2→ 2 H2O + O2                       (CAT: catalase)

In the organism the highly reactive (and thus toxic) hydroxyl radical can also be produced nonenzymatically through catalysis by transition metal ions like Fe2+and Cu+(the so-called Haber-Weiss- and Fenton reactions):

metal ions

H2O2+ O2• → OH• ++ O2                       (Haber-Weiss reaction)

H2O2+ Cu+/Fe2+→ OH• ++ Cu2+/Fe3+     (Fenton reaction)

In all likelihood the “full” Haber-Weiss reaction (i.e., the reduction of H2O2by O2•) is as follows (showing that the Fenton reaction is representing one particular part of the Haber-Weiss reaction):

O2• + Fe3+/Cu2+→ O2+ Fe2+/Cu+

H2O2+ Fe2+/Cu+→ OH• ++ Fe3+/Cu2+

Because iron is normally bound, free iron is maintained in the plasma at a very low level, and the cellular iron is not available to mediate a Fenton reaction in vivo. Biological reducing or chelating agents, or acidic pH, may however promote the release of iron from transport and storage proteins. Superoxide anion is transformed by superoxide dismutase to H2O2. Moreover, in the presence of traces of transition metal ions (iron salts may become available in vivo), superoxide anion and hydrogen peroxide undergo the so-called iron-catalyzed Haber-Weiss reaction which results in OH.formation. The hydroxyl radical is highly reactive and oxidises all organic chemicals, including biomolecules, when present in very close proximity to the place where the hydroxyl radical is formed. Superoxide and H2O2are less reactive and can diffuse away from their site of formation, leading to OH. generation whenever they meet a “spare” transition metal ion. H2O2also crosses all cell membranes easily. Thus, hydroxyl radicals are involved in H2O2related toxic effects. Oxygen radical formation can lead to lipid peroxidation, destruction of proteins, including enzyme inactivation, or to DNA damage.

A variety of in vitro cytotoxicity and genotoxicity studies with exogenous hydrogen peroxide indicate that chelation of iron dramatically decreases the toxic response thus demonstrating the important role of hydroxyl radical generation in toxicity under those conditions.

Genetic polymorphism of enzymes involved in detoxification:

In human populations there are genetically determined traits which determine the degradation capacity of hydrogen peroxide (catalase activity, level of reduced glutathione and hence the activity of the GSH redox cycle), notably in red blood cells. The distribution of blood catalase activity values was found to be trimodal, corresponding to the three phenotypes termed acatalasaemic, hypocatalasaemic and normal. Hypocatalasaemic individuals exhibited activities 36-55 per cent of the normal mean. About half of the individuals homozygous for acatalasaemia (blood catalase activities 0-3.2 per cent of normal) have clinical manifestations (Takahara's disease). In this disease, oral ulcerations develop mainly due to lack of catalase in blood and probably in tissues. Bacteria in the crevices of the teeth or tonsillar lacunas produce hydrogen peroxide. Since there is no catalase to decompose the H2O2 produced, haemoglobin is oxidised to methaemoglobin thus depriving the infected area of oxygen, and result in ulceration, necrosis and decay of the oral mucosa. In a Swiss population the frequency of homozygotes was about 0.04 per 1,000. The total number of reported patients of acatalasaemia worldwide (by 1989) was 107 belonging to 52 families. Acatalasaemia is assumed to be inherited as an incomplete autosomal recessive trait. Regarding Japanese acatalasaemia, the frequency of the recessive gene was estimated to be 0.00087, and the frequencies of heterozygotes and homozygotes were estimated to be 1.73x10-3and 4.23x10-6, respectively. In typical acatalasaemia, there is a trace of catalase activity in somatic cells; in atypical acatalasaemia, there is less catalase activity in blood cells (about 4% of normal) and a reduced activity in somatic cells. The frequency of hypocatalasaemia among Asian population averaged 0.2-0.4%; it was highest among Koreans (1.29%) and lowest among Japanese (0.23%). In Japanese groups comprising 4-5 individuals, the mean ± SD blood catalase activity was 3,380±180 Pu/g Hb among normal persons, 1,520±350Pu/g Hb for hypocatalasaemic cases, and 5.5±0.8 Pu/g Hb for acatalasaemic cases.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic disorder of erythrocytes (over 300 hundred variants have been identified) in which the inability of affected cells to maintain NAD(P)H levels sufficient for the reduction of oxidised glutathione results in inadequate detoxification of hydrogen peroxide through glutathione peroxidase. Presumably hydroxyl radicals from the peroxide damage the plasma membrane and the cells are prone to haemolysis. Haemolysis is often associated with the oxidation of haemoglobin to methaemoglobin and the formation of denatured haemoglobin (Heinz bodies). It is estimated that about 400 million people throughout the world are deficient in G6PD. Since the defective gene locus is on the X-chromosome, the enzymopathy is more common in males than in females. Prevalence rates vary from 63% among Kurdish Jews to very low rates of 0.1% or less in. G6PD dependent haemolysis and anaemia have become manifest on using pharmaceuticals which generate hydrogen peroxide in the human body (such as the antimalarial primaquine). However, only a fraction of the enzymopathic persons develop the syndrome.

Conclusions on toxicokinetics and metabolism of hydrogen peroxide:

Hydrogen peroxide is a normal metabolite in the aerobic cell, but there is uncertainty about the true levels of the substance in biological media due to analytical difficulties. The steady state level appears to depend on the balance between its generation and degradation. Hydrogen peroxide passes readily across biological membranes (permeability constant corresponds to that of water) and, because it slowly reacts with organic substrates, it can diffuse at considerable distances in the cell. There are two main hydrogen peroxide metabolising enzymes, catalase and glutathione peroxidase, which control H2O2concentration at different levels and in different parts of the cell as well as in the blood. At low physiological levels hydrogen peroxide is mainly decomposed by GSH peroxidase whereas the contribution of catalase increases with the increase of hydrogen peroxide concentration. Red blood cells remove hydrogen peroxide efficiently from the blood due to a very high catalase activity whereas in the serum catalase activity is low.

Both animal studies and human case reports indicate that at high uptake rates hydrogen peroxide passes the absorption surface entering the adjacent tissues and blood vessels where it is degraded liberating oxygen bubbles. One ml of 30 % H2O2yields approximately 100 ml of oxygen; thus mechanical pressure injury may be produced. The hazard of oxygen embolisation is particularly high if the substance is administered into closed body cavities where the liberated oxygen (under pressure) cannot freely escape. In most cases the consequences of venous embolisation are not catastrophic because the lung functions as an effective filter for microbubbles under normal conditions. However, in experiments with dogs, when the lungs were overloaded with a bolus injection of 30 ml of air, or when the animals were pretreated with a vasodilator (aminophylline) prior to venous infusion of microbubbles of air, embolisation was detected in the femoral artery with Doppler monitoring. Regarding hydrogen peroxide inhalation or skin contact at rates that would correspond to occupational exposures, there are no data on the systemic fate of the substance. In view of the high degradation capacity for hydrogen peroxide in blood it is however unlikely that the endogenous steady state level of the substance is affected. In biological systems, hydrogen peroxide may also undergo iron-catalyzed reactions (Fenton reaction, Haber-Weiss reaction) resulting in the formation of hydroxyl radicals. The cellular toxicity of hydrogen peroxide appears to depend largely on the generation of hydroxyl radicals. Genetically determined traits (acatalasaemia, glucose-6-phosphate dehydrogenase deficiency of the erythrocytes) render humans more susceptible to peroxide toxicity.

It was concluded that further scientific data are desirable on the toxicokinetics of hydrogen peroxide. After exploring the feasibility of such studies industry has concluded that presently it seems impossible to measure the fate of exogenous hydrogen peroxide as any measurement will interfere with physiological equilibria.

Conclusion:

The chemical nature of KMPS triple salt is characterised by oxidation at the site of first contact. As a consequence thereof, KMPS triple salt will rapidly degrade and will eventually form potassium and sulfate ions and hydrogen peroxide which are the relevant chemical entities to be considered for the assessment of the toxicokinetics and metabolism of KMPS triple salt. Based on these fundamental properties of KMPS triple salt, it wll be not bioavaiable, neither by inhalation, ingestion, or contact by skin. Bioaccumulation is unlikely.

 References

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