[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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 g
- 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 (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): 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): 12/12


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 (if gavage): 1 to 2 ml
- Lot/batch no. (if required): 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 other: mg/kg bw

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 d and 10 d 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 (delete / add / specify): urine
- Time and frequency of sampling: 24 hours after treatment.
- From how many animals: (samples pooled or not) Not clear 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.

Details on excretion:

The faeces was the major route of elimination (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 hr (urinary)

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 70 hr (urinary)

Test no.:

#1

Toxicokinetic parameters:

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

Test no.:

#1

Toxicokinetic parameters:

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

Metabolites identified:

yes

Details on metabolites:

URINARY METABOLITES (24 hr 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 toxicokinetic study, conducted in a similar manner to OECD 417 and in compliance with GLP, 150 mg/kg bw of 14C-labelled ATMP was administered to male Sprague-Dawley rats. It was concluded that ATMP was poorly absorbed and rapidly eliminated after oral administration. Bone was the only tissue that demonstrated significant accumulation of ATMP-derived radioactivity.

Reference 2

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

Application of radiolabelled substance and observation of absorption and excretion in urine.

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

5 male (bwt 208-222 g) and 5 female (171-215g) Wistar rats

Type of coverage:

occlusive

Vehicle:

water

Duration of exposure:

48 hours

Doses:

200 µl (0.2 ml) of a 0.88% aqueous solution (pH 7) of 14C-ATMP (equivalent to 1.76 mg ATMP/ rat; 27.1 µCi/rat) was applied to 10 cm² area of shaved skin (24 hours prior to application) under occlusion for 48 hours.
Comment: based upon a mean body weight of 216.4 g for males and 186.4 g for females, this was equivalent to 8.1 or 9.4 mg/kg bw, respectively.

No. of animals per group:

5 male, 5 female

Details on study design:

PREPARATION OF DOSING SOLUTION
The test sample was prepared by custom synthesis 8.50 mg 14C-ATMP (540 µCi) was mixed with 26.0 mg unlabelled material and 3954 mg double distilled water. Comment: the final concentration of ATMP in the dosing solution appears to be 0.89% and not 0.88% as stated in the report.

Comment: It is not clear how the solution was neutralised to pH 7.0; use of NaOH may have resulted information of the sodium salt.

SAMPLE COLLECTION
Radioactivity present in urine, faeces, exhaled air (14C-carbon dioxide), skin (application site), carcass and cage wash was quantified over a 48 hours post-application period.

Results presented as mean and SD.

Absorption in different matrices:

The majority of the dose remained at the application site 48 hours after application, with trace amounts excreted in urine, faeces and as 14C-carbon dioxide or remaining in the carcass:
- Urine: 0.2-0.5%
- Faeces: 0.087-0.03%
- CO2: 0.087-0.061%
- Skin (application site): 76.3-60.2%
- Carcass: 0.22-0.35% (Mean data for males or females, respectively) The total absorbed over 48 hours was 0.603% of the dose in males, and 0.940% of the dose in females.
Of the material absorbed:
- urinary excretion was virtually complete during the first 24 hours post-treatment (93-95% of total present in urine) with negligible excretion between 24-48 hours; - faecal elimination was comparable over 0-24 hr and 24-48 hours (approx. 50% of total excreted present at each time point);
- slightly more 14C-CO2 was exhaled over 0-8 hours (40-50% of total excreted) than over 8-24 hours or 24-48 hours.

Time point:

48 h

Dose:

200 µl of a 0.88% aqueous solution (pH 7) of 14C-ATMP

Parameter:

percentage

Remarks:

male rats

Absorption:

0.603 %

Time point:

48 h

Dose:

200 µl of a 0.88% aqueous solution (pH 7) of 14C-ATMP

Parameter:

percentage

Remarks:

female rats

Absorption:

0.94 %

absorption: 0.603% (male); 0.94% (female)

excretion: the majority of the absorbed radiolabelled ATMP was excreted in the urine within 24 hours of treatment.

Conclusions:

The cutaneous absorption of radiolabelled ATMP over 48 hours was 0.603% in male rats and 0.94% in females. 60-75% of the applied material was recovered from the application site at 48 hours. Absorbed. Radioactivity was excreted mainly in urine over the first 24 hours following application.

Description of key information

No toxicokinetics studies are available for ATMP-xNH4. An assessment has been made with reference to relevant chemical properties and read-across evidence from the Category member ATMP-H. See attachment to Section 13 for justification of read-across within ATMP Category.

 

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 (Monsanto, 1995).

 

In a good quality dermal absorption study, the cutaneous absorption of radiolabelled ATMP over 48 hours was 0.603% in male rats and 0.94% in females. 60-75% of the applied material was recovered from the application site at 48 hours. The absorbed radioactivity was excreted mainly in urine over the first 24 hours following application (Henkel, 1983b).

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

2.2

Absorption rate - dermal (%):

0.94

Additional information

No toxicokinetics studies are available for ATMP-xNH4. An assessment has been made with reference to relevant chemical properties and read-across evidence from the Category member ATMP-H. See attachment to Section 13 for justification of read-across within ATMP Category.

 

Additional information

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. There are no toxicokinetics data for ATMP-xNH4, therefore toxicokinetics data are read across from the Category member ATMP-H. It is not anticipated that the toxicokinetics of the ATMP salt will differ from the corresponding parent acid.

 

 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 Monsanto, 1995) and 2-10% in humans. Gastrointestinal pH is a major determinant influencing uptake and 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 1 - 2 at low pH (i.e. stomach) to 4 - 6 at more 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 pHs. 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 good quality dermal absorption study, the cutaneous absorption of radiolabelled ATMP over 48 hours was 0.603% in male rats and 0.94% in females. 60-75% of the applied material was recovered from the application site at 48 hours. The absorbed radioactivity was excreted mainly in urine over the first 24 hours following application (Henkel, 1983b). The low absorption rate can be explained by the hydrophilic characteristics of the substance.

 

 Inhalation

The vapour pressure of ATMP is extremely low (<10E-08 Pa). Consequently, inhalation of ATMP vapour is not possible. It is possible that a dust (from solid) or aerosol (from aqueous solution) of ATMP could be inhaled. The potential particle size distributions that workers and consumers could be exposed to for these forms of ATMP are currently not 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/day, and a 1211-fold increase after intravenous (i.v.) treatment with 15 mg/kg bw/day. Bone specificity of the substance is further supported in a study by Bartnik & Zimmerman (1983) following oral administration. Levels 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) (Monsanto, 1995).

Whole body autoradiography studies confirm the above tissue distribution findings, with pronounced deposition of 14C-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 day 10 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 (Monsanto, 1995).

 

 Metabolism

Unchanged ATMP accounts for 25% of material recovered from rat urine 0-24 hour after oral administration (150 mg/kg bw/day, 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 (Monsanto, 1995).

 

 Excretion

Faecal elimination of unabsorbed material predominates after ingestion (up to 90% of dose). Renal clearance of any 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, Monsanto (1995) demonstrated that faecal excretion was the principal route of elimination following gavage administration of 14C-ATMP to male rats (150 mg/kg bw/ day; 28.76 μCi/kg bw); 74% of the dose eliminated in 24 hours, 83% at 48 hours, up to a maximum 84% at 10 days. Trace amounts of radioactivity were present in urine (approximately 1% of dose) and blood, tissues and carcass (total approximately 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 hours post-dosing, rising to 50% after 24 hours (maximum 53% accounted for over 10 days). 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 (Monsanto, 1995).

 

 References

 Bartnik & Zimmerman (1983) Intestinale resorption von radioaktiv markiertem CA 3 nach oraler applikation an ratten