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The toxicokinetics of 4-Hydroxy TEMPO was assessed in a statement based on published data and the available toxicological profile. The test item is expected to be systemically absorbed after oral exposure and to diffuse into intracellular compartments. Distribution through extracellular body fluids is likely. Metabolism will most likely include microsomal monooxygenase reactions in the liver and will result in the quick formation of hydroxylamine metabolites. Excretion of the breakdown products will most likely occur via urine.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Toxicokinetics of 4-Hydroxy TEMPO

 

General

The following sections provide an overview of the toxicological profile of 4-Hydroxy TEMPO, and an analysis of the substances toxicokinetic behaviour. Belonging to the group of nitroxyl radicals, it is generally accepted that bio reduction to hydroxylamines represents the main metabolic pathway in mammals. In addition, other metabolites such as secondary amines, ammonium cations, radical adducts or other derivatives may be formed (Kroll & Borchert, 1999). It is assumed that nitroxides follow double exponential decay kinetics, with a fast and a slow component. The fast component resulting from nitroxide distribution is in the order of seconds followed by nitroxide metabolism and clearance in the order of minutes.

 

 

Toxicological profile

In an acute oral toxicity study (CIBA GEYGY Ltd., 1983, Report No. TK 11725/2) according to OECD test guideline 401, a single oral dose of 4-Hydroxy TEMPO was administered to 5 rats per gender in distilled water at doses of 0, 500, 1000, 2000 or 5000 mg/kg bw. Animals were observed for 14 days and then necropsied. Fatalities occurring at 500 mg/kg bw: 0 males and 0 females; 1000 mg/kg bw: 3 males and 1 female; 2000 mg/kg bw: 5 males and 5 females; 5000 mg/kg bw: 5 males and 5 females. Surviving animals recovered within 9 days. The following treatment related clinical signs were noted: Dyspnoea, exophthalmus, ruffled fur and curved body position. In addition: Ventral and lateral body position, tonic-clonic convulsions and sedation (in all dose groups: dyspnoea and convulsions, the latter for up to 5 hours past dose, incl. non-moribund animals). The combined oral LD50was 1053 mg/kg bw (males: 953; females 1155 mg/kg bw). The study was QA-audited (GLP was not compulsory in 1983). Based on the results of the acute oral toxicity study, the test substance is classified as Xn, R22 Harmful (Harmful if swallowed) according to the criteria of EU Directive 67/548/EEC and as Cat. 4, H302 (Harmful if swallowed) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008

A limit-dose study (2012-0068-DGT) of dermal toxicity was conducted according to OECD TG 402, Regulation (EC) No 440/2008 method B.3 and OPPTS 870.1200. A dose of 2000 mg/kg bw of 4-Hydroxy TEMPO in distilled water was applied on rats (5/sex) once for 24 hours on the shaved intact dorsal skin. The test patch was occlusive. This treatment was followed by an observation period of 2 weeks. No influence on animal behaviour or premature death was noted. The body weight gain was not influenced by the test item administration. No skin reactions were observed at the application site. The macroscopic examination did not reveal any changes. The LD50 could not be calculated due to lack of death at the applied dose level. Thus, the Dermal LD50 was determined to be: >2000 mg/kg bw.

 

In a GLP-compliant OECD test guideline 404 study (Hüls AG 1995, AH-95/0151), three male Russian white rabbits were treated dermally with 0.5 g 4-Hydroxy TEMPO moistened with 0.5 cm³ water using semi occlusive conditions for 4 hours. An application site of approximately 6 cm² was covered with the test substance. The animals were observed for 72 hours and skin changes were recorded and scored for every animal after 30 to 60 minutes, 24, 48 and 72 hours. The application of the test substance to the skin caused a slight edema in one rabbit and a slight erythema in all three rabbits. Signs of edema and erythema fully reversed within 72 hours at the latest. No signs of systemic toxicity were observed. The individual average scores (24 to 72 h) for irritation did not warrant a classification according to EU criteria.

In a GLP-compliant OECD test guideline 405 study (Hüls AG 1995, AA-95/0151), the right eye of three male Russian white rabbits was exposed to 0.1 g 4-Hydroxy TEMPO, placed as dry substance into the conjunctival sac of the eyelid. Animals were observed after 1, 24, 48 and 72 hour(s) after treatment and 6, 8, 10, 13, 16, 21 and 24 days afterwards. The eyes were washed out with physiological saline solution after 24 hours. Overall, the treatment caused opacity and conjunctivae/chemosis of the eye mucous membranes as well as exsudation and detachment of upper layers of mucosa at 48 hours. In 2 of 3 animals the effects were slowly reversible after 13 to 21 days. In one animal hyperaemia, swelling of the conjunctivae and corneal opacity persisted up to day 24, when the study was terminated. Due to the individual average scores (24 to 72 hours) of conjunctivae, chemosis, opacity and iritis, the test substance was graded as irritant under the conditions of this study. The test substance is classified as Xi, R41 (Irritant, Risk of serious damage to eyes) according to the criteria of EU Directive 67/548/EEC and as Cat. 1, H318 (causes serious eye damage) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.

In a skin sensitisation assay (Bühler test: Hüls AG 1995, HS-95/0151), performed according to GLP and the requirements of OECD test guideline 406, 20 female Guinea pigs received three dermal doses (day 0, 7 and 14) of 0.5 g 4-Hydroxy TEMPO as a paste in petrolatum at a 50 % (w/w) concentration onto their left caudal flank, while nine female negative control animals received the vehicle alone. The maximum concentration feasible was determined in a pre-test. The animals were challenged on day 28, using the same vehicle and test item concentration, applied onto the right flank. No signs of skin sensitisation where detected. The test item is not classified with regard to skin sensitisation according to Directive 67/548/EEC and Regulation (EC) No 1272/2008 (GHS, CLP).

In a repeated dose toxicity study according to GLP and OECD Guideline 407 (Exxon Chem Co, 1992,N88-930000244), the test item in water was administered orally by gavage to six SD-rats per dose group and gender at 8, 40, 200 or 1000 mg/kg bw/day for 28 days. The control group received the vehicle alone. Additional recovery groups of the same size, comprising vehicle and high dose animals, were monitored for further 14 days to assess reversibility and delayed onset of test item related effects. There were no fatalities; body weight, food consumption, serum chemistry and urinalysis parameters remained unaffected. Apart from salivation, which was mainly observed in all high dose animals, no abnormal clinical signs occurred. The most prominent effects in the high dose groups were a decreased red blood cell count, haemoglobin and haematocrit and an increase of reticulocytes. Low corpuscular haemoglobin concentrations and high counts of segmental neutrophils were found in high dose females only. The decrease of red blood cell counts and haemoglobin concentration persisted throughout the recovery phase. Absolute and relative spleen and liver weights were increased in high dose animals of both sexes at the end of the dosing period, spleen weight reduction persisted throughout the recovery phase. High dose animals showed a reversible blackening of spleen hue. Histopathology revealed congestion and hemosiderin accumulation in the spleen of all high dose animals and the females of the 200 mg/kg bw/d group. In addition, swelling of hepatocytes was noted in all high dose animals. All histopathological effects were detectable until the end of the recovery phase. Based on these findings, the liver and spleen were identified as the target organs. The NOAEL was set to 40 mg/kg bw/day.Based on the results of the subacute oral toxicity study, the test substance was classified as STOT RE cat 2 with H373 (May cause damage to organs through prolonged or repeated exposure) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.

The genetic toxicity of 4-Hydroxy TEMPO was tested in a GLP compliant OECD 471 guideline study (Ames test, classification: weakly mutagenic with metabolic activation), and in a GLP compliant OECD 476 guideline study (In vitroMammalian Cell Gene Mutation Test, classification: not mutagenic). In addition, the genetic toxicityin vivoof 4-Hydroxy TEMPO was tested in a GLP compliant OECD 474 guideline study (mouse micronucleus test, classification: not mutagenic) and in a GLP compliant OECD 486 guideline study (unscheduled DNA synthesis, classification: not mutagenic). Although Ames test results indicate a weak genotoxicity potential in the presence of a rat S9-mixin vitro, 4-Hydroxy TEMPO is not subject to classification for mutagenicity according to EU Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008 due to the unanimously negative results in the HPRT assay as well as the negative results of thein vivoassays (mouse micronucleus test and unscheduled DNA synthesis).

 

Toxicokinetic analysis of 4-Hydroxy TEMPO

 

4-Hydroxy TEMPO is solid at room temperature with a molecular weight of 172.24 and an absolute density of 1.127 g/cm³ at 20°C. The substance has a melting point at 70°C and a boiling point at 332.21°C. Water solubility of 4-Hydroxy TEMPO, calculated from the logPowof 0.56, was estimated to be 19.6 g/L. The vapour pressure was determined to be 1.48-6Pa (25°C).

 

Absorption:

Based on molecular weight, logPowand water solubility, 4-Hydroxy TEMPO is likely to be absorbed in the GI tract. The positive logPowof 0.56 indicates that the substance is rather lipophilic and therefore in the range between -1 and 4, where it will diffuse well across plasma membranes. In addition, gastro-intestinal absorption of 4-Hydroxy TEMPO is triggered by passage through aqueous pores or carriage with the bulk passage of water, which is favoured for small water soluble substances (molecular weight < 200 g/mol). It is unclear whether an active transport for 4-Hydroxy TEMPO exists. Furthermore, oral absorption may be limited by microbiological degradation of the intestinal microflora. Nevertheless, extensive gastrointestinal absorption is expected for 4-Hydroxy TEMPO based on its physicochemical properties.

 

Due to the very low vapour pressure of 1.48 x 10-6Pa, 4-Hydroxy TEMPO does not vaporise sufficiently to become available for an inhalation exposure of relevance. Exposure to aerosol from solids is highly unlikely. However, due to the relatively high water solubility, dusted substance may partly dissolve on the mucous membranes of the respiratory tract, but the extent of subsequent systemic absorption is probably negligible. The substance characteristics and physicochemical properties indicate that dermal absorption is not a major route of exposure, as the solid state favours slow dermal absorption with liquids taken up more readily than dry particulates. Similar to a postulated inhalation exposure, it is unlikely that systemic dermal absorption from dusted particles in sweat will attain relevant quantities.

 

Distribution:

Based on the physicochemical properties (molecular weight: 172.24, logPow: 0.56, water solubility: 19.6 g/L), 4-Hydroxy TEMPO is likely to be distributed systemically throughout the extracellular compartments. This assumption is supported byin vivodata (Coveyet al., 2000); where after ani.v.application to rats, the maximum plasma concentration of 4-Hydroxy TEMPO was reached after 5 min and amounted to 62.2 µg/ml, with a large mean volume of distribution at steady state of 4513 ml. The concentration declined bi-exponentially with an initial half life of 17 min and a terminal half life of 10 h. 4-Hydroxy TEMPO was still measurable at 24 hrs. Plasma protein binding was approximately 20% (0.1 - 1 µg/ml). Data obtained from oral sub chronic repeated dose toxicity testing (Exxon Chem Co, 1992,N88-930000244) identified liver and spleen as target organs. The low half life, based on the presumed rapid metabolic degradation and subsequent quick urinary excretion, indicate a low potential for bioaccumulation.

 

It can be assumed that distribution of the parent compound is limited through the dermal route, because 4-Hydroxy TEMPO was shown to be rapidly metabolised in a human keratinocyte cell line (Krollet al., 1999).

 

 

Metabolism:

As 4-Hydroxy TEMPO was rapidly metabolised in hepatocyte-cultures (Iannoneet al., 1989) with a constant rate of Nitroxyl radical depletion at 3.59 x 10-2, it may be concluded that 4-Hydroxy TEMPO undergoes a first pass effect in liver after oral application. Two metabolites were identified (Kroll et al., 1999) by GC, GC-MS, the corresponding hydroxylamine and the secondary amine. After i.v. application to rats (Coveyet al., 2000) the hydroxylamine metabolite was detected already after 5 min (28.4 µL/ml) and declined in a bi-exponential manner, similar to the parent compound. The initial half life was 15 min and the mean apparent terminal half life was 8.4 h. The mean AUC value of the hydroxylamine was 51.5 µg/ml/h, 1.28 fold that of 4-Hydroxy TEMPO (40.2 µg/ml/h), supporting the assumption that nitroxide metabolism and clearance occurs in the order of minutes. Of the phase I & II enzymes investigatedin vivo, neither Cytochrome P450 nor glucoronidase / arylsulfatase had an impact on carbonyl reduction of the structurally closely related TEMPONE in perfused rat liver (Kroll & Borchert, 1999). 4-Hydroxy TEMPO was shown to be rapidly metabolised in a human keratinocyte cell line (Krollet al., 1999), thus it can be concluded that 4-Hydroxy TEMPO undergoes cellular metabolism (hydroxylamine formation) after dermal as well as oral exposure.

 

No conversion into a metabolite that is more toxic than the parent is expected as indicated byin vivorepeated dose and genetic toxicity tests performed with 4-Hydroxy TEMPO.

 

Excretion

According to Coveyet al.(2000), the mean clearance was 841.3 ml/min. The relatively low molecular weight of 172.24, a relatively high water solubility of 19.6 g/L and the observation that neither phase I nor phase II enzymes seem to be involved in the metabolism of 4-Hydroxy TEMPO suggest that the substance and the main metabolites (hydroxylamine and the secondary amine) are rapidly excreted via urine. If administered orally, excretion may also occur via faeces as metabolic degradation via the intestinal micro flora is expected.

 

Summary

4-Hydroxy TEMPO is expected to be systemically absorbed after oral exposure. Based on its physicochemical properties, the substance is expected to diffuse into intracellular compartments. Distribution through extracellular body fluids is likely. Metabolism will most likely include microsomal monooxygenase reactions in the liver and will result in the quick formation of hydroxylamine metabolites. Excretion of the breakdown products will most likely occur via urine. No bioaccumulation is expected.

 



References

CIBA-GEIGY-Ltd. 1983, Acute Oral LD50 in the Rat, Study Report No. TK 11725/2, TSCA Section 8e submission, 1992, Doc. 88-920001983, NTIS/OTS0536225

 

Covey, J.M., Chan, K.K., Chiu M. (2000). Pharmacokinetics of TEMPOL and reduced TEMPOL in the rat as measured by a specific GC/MS assay. Pharmacol. Exp. Ther. 51, 705.

 

Exxon Chem Co. (1992), 28 day repeated-dose oral toxicity study of 4-Hydroxy TEMPO in rats. Study Report N88-930000244: TSCA Sect. 8e submission, OTS0545601.

 

Hüls AG 1995, Prüfung auf akute Hautreizwirkung von 4-OH-TEMPO am Kaninchen (Test of acute skin irritation of 4-OH-TEMPO in the rabbit), Study Report No.AA-95/0151

 

Hüls AG 1995, Prüfung auf akute Augen- und Schleimhautreizung von 4-OH-TEMPO am Kaninchen (Acute test of eye and mucosal irritation of 4-OH-TEMPO in the rabbit), Study Report No.AA-95/0151

 

Hüls AG 1995, Prüfung auf Sensibilisierung der Haut von 4-OH-TEMPO am Meerschweinchen (Test of sensitising properties of 4-OH-TEMPO in the Guinea pig), Study Report No.HS-95/0151

 

Iannone, A., Hu, H., Tomasi, A., Vannini, V., Swartz, H.M. (1989).Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen and structure of nitroxides. Biochim. Biophys. Acta, 991, 90-96.

 

Kroll, C., Borchert, H.H. (1999), Metabolism of the stable nitroxyl radical 4-oxo-2,2,6,6,-tetramethylpiperidine-N-oxyl (TEMPONE). Eur. J. Pharm. Sci., 8, 5-9.

Kroll, C., Langner, A., Borchert, H.H. (1999), Nitroxide metabolism in the human keratinocyte cell line HaCaT. Free Rad. Biol. and Med., 26 (7/8), 850-857

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