Pentapotassium ({[(hydroxyphosphinato)methyl](phosphonatomethyl)nitroryl}methyl)phosphonate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.11.1991 to 16.07.1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Only one dose tested.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Portage or Charles River Kingston
- Age at study initiation: 51 to 81 days
- Weight at study initiation: 242 to 393 grams
- Fasting period before study: No data
- Housing: Individual stainless steel cages with wire mesh bottoms, or Roth-type metabolism cages.
- Individual metabolism cages: yes
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 14-31 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light


IN-LIFE DATES: From: 27.12.1991 To: 16.07.1993

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in distilled water. All dosing solutions were aliquoted by weight, diluted and weighed portions were counted by Liquid Scintillation Counting to determine concentration and specific activity.


VEHICLE : Distilled water
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: No data
- Amount of vehicle: 1 to 2 ml
- Lot/batch no.: No data
- Purity: No data


HOMOGENEITY AND STABILITY OF TEST MATERIAL: No data

Duration and frequency of treatment / exposure:

Single dose (animals killed ten days later)

Dose / conc.:

150 mg/kg bw/day

Remarks:

Received dose was calculated by weighing syringe plus dosing needle before and after use. Specific activity of dosing solution = 1073600 dpm/mg; equivalent amount of radioactivity = 28.76 uCi/rat.

No. of animals per sex per dose / concentration:

Males: four
A further two rats (dosed as above) were sacrificed 1 day and 10 day post-treatment for whole-body autoradiography. 
A further two rats were dosed as above and were sacrificed after 72 hours. This group was used to determine whether radioactivity was present in expired CO2, but was not discussed in the study report.

Control animals:

no

Positive control reference chemical:

None

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): No data

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood, tissues, cage washes
- Time and frequency of sampling: Urine, faeces and cage washes at 24 hour intervals after dosing until sacrifice, in-life blood samples were collected at 15 and 30 minutes, 1, 2, 4, 6, 12 and 24 hours after dosing, then daily thereafter. At sacrifice blood samples were obtained and the following tissues and organs obtained: liver, kidneys, bone (femur), spleen, skeletal muscle, bone marrow. Gastrointestinal contents were collected by flushing the intestinal tract with saline. All samples were stored frozen at -20oC.


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: 24 hours after treatment.
- From how many animals: Unclear from study report
- Method type(s) for identification: HPLC
- Limits of detection and quantification: No data

Statistics:

Data were presented as the mean ± the standard error of the mean (SEM).

Details on absorption:

The percent of ATMP absorbed was calculated to be approximately 2.2 %.
Urine = 1.1% Cage wash = 0.305% Carcass = 0.209% Tissue/organs = 0.055% Gut contents = 0.022% Blood = 0.0006% Plasma = 0.0005%

Details on distribution in tissues:

Approximately 0.2 % of the dose was found in the carcass of animals dosed orally. Very little ATMP-derived radioactivity remained in any of the other analysed tissues ten days after dosing. When comparing the tissue to blood levels (see Table 2), the bones had the highest tissue to blood ratios.

Observation:

not determined

Details on excretion:

The major route of elimination was by faeces (84%), while urine (1.1%) contributed much less (see Table 1). No significant amount of 14C was present in exhaled carbon dioxide (no further details). 

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 5 hours (urinary)

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 70 hours (urinary)

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 5.3 hours (whole body)

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 299 hours (whole body)

Metabolites identified:

yes

Details on metabolites:

URINARY METABOLITES (24 hours sample) Parent compound = 25.1%, N-methyl derivative = 45.9%; Unidentified = 28.6% (no further details)

Table 1 Summary of urinary and faecal elimination.

Day	Faecal elimination (%)	Urinary elimination (%)
1	84.32	1.016
2	9.448	0.056
3	0.34	0.019
4	0.031	0.013
5 -10	0.242	0.0342

Table 2 Summary of blood:tissue ratios at Day 10

Tissue	Ratio
Tibia	191
Femur	158
Bone marrow	104
Sternum	75.6
Carcass	7.81
Gut contents	3.63
Kidneys	2.61
Spleen	1.8
Liver	1.1
Blood	1.0
Erythrocytes	0.792
Muscle	0.644
Plasma	0.02

RECOVERY DATA
Individual total recovery = 81.85 - 88.55%
Mean total recovery = 85.90% (SEM = 1.62)

AUTORADIOGRAPHY
At 24 h, the major regions of localisation of 14C were:
- gut contents
- stomach contents
- nasal turbinates
- bone and bone marrow
Radioactivity also observed in the kidney (no other tissues) and throughout all bones of the body (most intense in epiphyseal plate of the long bones and in nasal turbinates).

At 10 d post-dose, intense localisation still apparent in bone, especially the epiphyseal plate of the long bones. Some low level deposition of 14C was present in stomach lining and the kidneys (no other tissues affected). (The authors note that this pattern is consistent with that reported for EHDP.)

Conclusions:

In a well conducted toxicokinetics study, conducted according to OECD TG 417 and in compliance with GLP, ATMP was poorly absorbed and rapidly eliminated after oral administration. Total recovery was 86%. The majority of the dose (84.2%) was excreted in the faeces and only 1.1% was excreted in the urine. The amount of radioactivity in the urine was used to determine the extent of absorption by comparing urinary excretion between orally and intravenously dosed rats. Using this approach, absorption following administration of 14C-labelled ATMP was shown to be approximately 2.2%. The initial and terminal urinary half-lives were approximately 5 and 70 hours, respectively. The initial phase whole-body elimination half-life was 5.3 hours and the terminal half-life was 299 hours. HPLC analysis of urine samples collected 24 hours after administration revealed the presence of the parent compound, the n-methyl derivative and an unidentified metabolite. Approximately 0.06% of the dose was found in the bone (femur, tibia and sternum) and 0.21% of the dose was found in the carcass. The overall tissue distribution confirmed that the highest levels of radioactivity were in the bone. The only tissue that demonstrated significant accumulation of ATMP-derived radioactivity was bone.

Description of key information

In a well conducted toxicokinetics study, conducted according to OECD Test Guideline 417 and in compliance with GLP, ATMP was poorly absorbed and rapidly eliminated after oral administration. Total recovery was 86%. The majority of the dose (84.2%) was excreted in the faeces and only 1.1% was excreted in the urine. The amount of radioactivity in the urine was used to determine the extent of absorption by comparing urinary excretion between orally and intravenously dosed rats. Using this approach, absorption following administration of ¹⁴C-labelled ATMP was shown to be approximately 2.2%. The initial and terminal urinary half-lives were approximately 5 and 70 hours, respectively. The initial phase whole-body elimination half-life was 5.3 hours and the terminal half-life was 299 hours. HPLC analysis of urine samples collected 24 hours after administration revealed the presence of the parent compound, the n-methyl derivative and an unidentified metabolite. Approximately 0.06% of the dose was found in the bone (femur, tibia and sternum) and 0.21% of the dose was found in the carcass. The overall tissue distribution confirmed that the highest levels of radioactivity were in the bone. The only tissue that demonstrated significant accumulation of ATMP-derived radioactivity was bone (Holt et al., 1995).

Key value for chemical safety assessment

Absorption rate - oral (%):

2.2

Additional information

ATMP-N-oxide is an analogue of ATMP that is more polar but has very similar metal-chelating properties. The high polarity and metal-complexing capacity of ATMP are responsible for their environmental and toxicokinetic behaviour: viz, irreversible binding onto the mineral fraction of soils and sediments, bioaccumulation not predicted by the log K_ow, poor adsorption from the gut and skin and partitioning into bone when administered to animals. The similar properties of ATMP-N-oxide would predict similar environmental and toxicokinetic behaviour i.e. these properties can be read-across from ATMP.

The sodium and potassium salts of ATMP-N-oxide can be considered in the same way because they would dissociate to the relevant phosphonate anions and K⁺or Na⁺, which would not be toxicologically significant in the context of chemical safety assessment.

The two phosphonate structures (ATMP and ATMP-N-oxide) can be considered to be a pair of structurally-analogous substances, within which many properties are generally consistent but in general do not follow predictable trends.

Introduction

Based on the available data, no major differences appear to exist between animals and humans with regard to the absorption, distribution and elimination of phosphonic acid compounds in vivo. Unless otherwise stated, the following information comes from the SIDS Initial Assessment Report for SIAM 18. Category: Phosphonic Acid Compounds Group 1. Annex VI.

Commercial formulations vary in concentration between suppliers and formulation types. The pH value of solutions at around 1% in water are shown in Table 1 below. These values are relevant to the various irritation and acute studies. Further company-confidential data are available if necessary.  

Table1: pH values of aqueous solutions

CAS	Product at typically 1%	Status
6419-19-8 (ATMP-H)	pH <2	Acid
94021-23-5 (ATMP-4Na)	pH ~7	Salt
2235-43-0 (Me-ATMP-5Na)	pH ~11	Salt

The above information is relevant to several of the human health end points.

ATMP salts are freely soluble in water and, therefore, the ATMP anion is fully dissociated from its potassium or sodium cations when in solution. The ATMP anion has six P-OH groups that can be ionised. They lose a hydrogen to form a negatively charged group (P-O^-). Under any given conditions, the degree of ionisation of the ATMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was ATMP-xNa, ATMP-xK, ATMP-H, or another salt of ATMP. Hence, some properties for a salt (in contact with water or in aqueous media) can be directly read across (with suitable mass correction) to the parent acid and vice versa. In the present context the effect of the alkaline metal counter-ion (sodium/potassium) will not be significant and has been extensively discussed in the public literature. In biological systems and the environment, polyvalent metal ions will be present, and the phosphonate ions show very strong affinity to them. Therefore, read-across within the ATMP category is considered appropriate. 

Absorption

Oral

The physicochemical properties of phosphonic acid compounds, notably their high polarity, charge and complexing power, suggests that they will not be readily absorbed from the gastrointestinal tract. This is supported by experimental data which confirm that absorption after oral exposure is low, averaging 2-7% in animals (2.2 % in Ceregen, Monsanto Company, 1995) and 2-10% in humans. Gastrointestinal pH is a major determinant influencing uptake. It is relatively acidic in the stomach (range: pH 1 - 4) and slightly more alkaline in the intestine (pH 4 - 7). The number of ionisations of the phosphonic acid moiety increases with increasing pH, rising from 0 - 1 at low pH (i.e. stomach) to 4 at neutral pH (reflective of conditions in the intestine). The negative charge on each molecule also increases with each ionisation, further reducing the already low potential for uptake. Stability constants for the interaction of phosphonic acids with divalent metal ions are high, and indicate strong binding, especially at lower pH. Complexation of a metal with a phosphonic acid would produce an ion pair of charge close to neutral which might favour absorption; however, the overall polarity of the complex would remain high thereby counteracting this potential. Overall, these considerations indicate that ingested phosphonic acid compounds will be retained within the gut lumen.  

 

Dermal

ATMP is too hydrophilic to be absorbed through the skin. In a dermal absorption study, 24-hour application of radiolabelled, neutralised ATMP-H onto the skin of rats, resulted in dermal absorption of 0.603% in male rats and 0.94% in female rats (Henkel, 1983).

 

Inhalation

The vapour pressure of ATMP is extremely low (<10E-08 Pa). Consequently, inhalation of ATMP vapour is not possible. It is possible that aerosol (from aqueous solution) of ATMP could be inhaled. The potential particle size distributions that workers and consumers could be exposed to is not currently known. However, the very high water solubility of this substance suggests that absorption will be low.  

 

Distribution

Blood / tissue ratios demonstrate that ATMP has a strong affinity for bone, with a 158-fold increase of 14C present in femur (relative to that in blood) following gavage administration of 150 mg/kg bw ATMP-H, and a 1211-fold increase after intravenous (i.v.) treatment with 15 mg/kg bw ATMP-H. Bone specificity of the substance is further supported in a study by Henkel (1983) following oral administration. Levels of ATMP-H were also increased in tibia (191-fold) and sternum (76-fold) after oral (gavage) treatment (not determined following injection). In contrast, amounts present in soft tissue (e.g. liver, kidney, spleen) and carcass were largely unaltered after gavage exposure (increase 8-fold or less) while i.v. injection was associated with greater increases (soft tissues elevated 3 to 30 fold; carcass 50 fold) (Ceregen, Monsanto Company, 1995). Whole body autoradiography studies confirm the above tissue distribution findings, with pronounced deposition of14C-ATMP (150 mg/kg bw, by gavage) in the epiphyseal plate of the long bones and also the nasal turbinates, with additional radioactivity present in gut contents and bone marrow. By 10 days post-treatment, intense localisation of label was still apparent in the epiphyseal plate of the long bones, with some material present also in stomach lining and kidneys (Ceregen, Monsanto Company, 1995).  

 

Metabolism

Unchanged ATMP accounts for 25% of material recovered from rat urine 0-24 hr after oral administration (150 mg/kg bw, by gavage), with 46% present as an N-methyl derivative and 29% as an unknown metabolite. In contrast, the parent substance predominated (64% of total) in urine after i.v. dosing (15 mg/kg bw), with approximately equivalent amounts of the N-methyl derivative (21%) and the unknown metabolite (14%) also present (Ceregen, Monsanto Company, 1995).  

There is evidence in the scientific literature for the metabolic inter-conversion of amines and their N-oxides. This takes place principally in the liver but also at lower rates in other tissues (Sugiura et al., 1976). N-oxides are common metabolites of tertiary amine drugs; examples are zolmitriptan (Wild et al., 1999), nicotine, where the pyrrole N-oxide is a major metabolite (Gorrod and Wahren, 1993) and morphine (Yeh et al., 1993). Examples of the metabolic reduction of amine N-oxides to amines are the veterinary antimicrobial olaquindox (Liu et al., 2010) and imipramine N-oxide (Kitamura and Tatsumi, 1997). In the liver, oxidation of amines to N-oxides and reduction of N-oxides to amines are both catalysed by the cytochrome P450 complex with the hydrogen donor being NADPH in the case of N-oxide reduction (Sugiura et al., 1976). This apparent contradiction is due to the presence of multiple isoforms of cytochrome P450 with different catalytic specificities expressed in mitochondria and endoplasmic reticulum (Wild et al., 1999). Where the tertiary amine is the active drug, it is usually found that its N-oxide is less active. An example is morphine-N-oxide, which is 11-89 fold less active as an analgesic than morphine (Fennessy, 1968), although the N-oxide reductase pathway complicates the analysis, as the N-oxide may be inactive per se and the lesser activity seen in vivo may be due to metabolic conversion.

 

Excretion

Faecal elimination of unabsorbed material predominates after ingestion (up to 90% of dose). Renal clearance of test material absorbed from the gut is rapid, with urinary half-lives of 5 hours and 70 hours reported. This second phase of excretion may represent mobilisation of material initially sequestered by bone, since deposition studies have shown preferential accumulation of these substances in the epiphyseal plate and other regions of the long bones in vivo. 

 

In a well-designed and reported study, Ceregen, Monsanto Company (1995) demonstrated that faecal excretion was the principal route of elimination following gavage administration of14C-ATMP to male rats (150 mg/kg bw; 28.76 μCi/kg bw); 74% of the dose eliminated in 24 hr, 83% at 48 hr, up to a maximum 84% at 10 d. Trace amounts of radioactivity were present in urine (approx. 1% of dose) and blood, tissues and carcass (total approx. 0.3%) but not in exhaled air. Overall mean recovery from all sources was 85.9%. In contrast, renal clearance predominated after i.v. injection (15 mg/kg bw; 1.93 μCi/kg bw), with 46% of the dose recovered in urine 6 hr post-dosing, rising to 50% after 24 hr (maximum 53% accounted for over 10 d). Overall mean recovery was 88.9%. Approximately 4 to 5% of the dose was eliminated via faeces, while blood, tissues and carcass contained a total of 23% of the dose. Based upon relative urinary excretion after gavage and i.v. administration, gastrointestinal uptake was calculated as 2.15%. Kinetic analyses indicate that ATMP is excreted in a biexponential manner by the rat, with urinary half-lives of 5 hours or 70 hours after oral exposure, and 2 hours or 127 hours after i.v. treatment (Ceregen, Monsanto Company, 1995).

References:

Fennessy, M.R. (1968) The analgesic action of morphine-N-oxide Br. J. Pharmacol. 34: 337-344.

Gorrod, J.W. and Wahren, J. (eds) Nicotine and Related Alkaloids: Absorption, Distribution, Metabolism and Excretion Chapman & Hall, London, 1993

Liu, Z.Y., Huang, L.L., Chen, D.M., Dai, M.H., Tao, Y.F., Yuan, Z.H. (2010) The metabolism and N-oxide reduction of olaquindox in liver preparations of rats, pigs and chicken Toxicol Lett. 195: 51-59

Kitamura, S., Tatsumi, K. (1997) Menadione-dependent reduction of tertiary amine N-oxide by rat liver cytosol Biochem. Mol. Biol. Int. 42: 271-276

Sugiura, M., Iwasak, K., Kato, R. (1976) Reduction of Tertiary Amine N-Oxides by Liver Microsomal Cytochrome P-450 Mol. Pharmacol. 12: 322-334

Wild, M.J., McKillop, D., Butters, C.J, (1999) Determination of the human cytochrome P450 isoforms involved in the metabolism of zolmitriptan. Xenobiotica. 29: 847-857

Yeh, S.Y., Krebs, H.A., Gorodetzky, C.W. (1979) Isolation and identification of morphine N-oxide alpha- and beta-dihydromorphines, beta- or gamma-isomorphine, and hydroxylated morphine as morphine metabolites in several mammalian species. J Pharm Sci. 68: 133-140