Reaction mass of iron chelate of sodium salt N-[1,2 dicarboxyethyl] D,L aspartic acid and sodium chloride

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

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

Adequacy of study:

key study

Study period:

2006-10-16 to 2006-12-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EU Council Directive 91/414/EEC amended by the Commission Directive 94/79/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen (Aktenzeichen: VI-3-31.11.62.04)

Test material

Test material information

Constituent 1

Reference substance name:

144538-83-0

Cas Number:

144538-83-0

IUPAC Name:

144538-83-0

Constituent 2

Reference substance name:

2-(1,2-Dicarboxy-ethylamino)-succinic-acid tetra- Na-salt

IUPAC Name:

2-(1,2-Dicarboxy-ethylamino)-succinic-acid tetra- Na-salt

Constituent 3

Reference substance name:

Imminodisuccinic acid, tetrasodium salt

IUPAC Name:

Imminodisuccinic acid, tetrasodium salt

Test material form:

other: aqueous solution

Details on test material:

- Name of test material (as cited in study report): Iminodisuccinate, Na4 salt (Baypure® CX100)
- Molecular formula (if other than submission substance): C8H7NNa408
- Molecular weight (if other than submission substance): 337 g/mol
- Smiles notation (if other than submission substance): C(C(C(=O)[O-])NC(CC(=O)[O-])C(=O)[O-])C(=O)[O-].[Na+].[Na+].[Na+].[Na+]
- InChl (if other than submission substance): InChI=1S/C8H11NO8.4Na/c10-5(11)1-3(7(14)15)9-4(8(16)17)2-6(12)13;;;;/h3-4,9H,1-2H2,(H,10,11)(H,12,13)(H,14,15)(H,16,17);;;;/q;4*+1/p-4
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: chelating agent
- Physical state: 14C (aqueous solution), 13C (solid material), unlabelled (aqueous solution)
- Composition of test material: 14C (Diastereomer ratio: 49.1 / 50.9 (HPLC, 14C-detector)); 13C (51.4 / 48.6 (13C-NMR)); unlabelled (52.1% (mesonics:R,S-IDS) / 47.9% (enantiomerics: R, R or S,S-IDS))
- Expiration date of the lot/batch: unlabelled (2007-03-15)
- Radiochemical purity (if radiolabelling): 14C: > 97%; 13C: 99.5% (NMR)
- Specific activity (if radiolabelling): 14C: 4.31 MBq/mg = 116.5 µCi/mg
- Locations of the label (if radiolabelling): [14C]label positions: 2,3-14C1, 2'3'-14C1; [13C]label positions: 2,3-13C1, 2'3'-13C1 (see figure attached: + points to the labelled C atoms)
- Expiration date of radiochemical substance (if radiolabelling): no data
- Stability under test conditions: not reported
- Storage condition of test material: not reported
- Other: Concentration of stock solution: 14C (3.66 mg IDS/g solution); 13C (102.7 mg IDS/g solution); unlabelled (347 mg IDS/g solution)

Radiolabelling:

yes

Remarks:

[14C]label positions: 2,3-14C1, 2'3'-14C1; [13C]label positions: 2,3-13C1, 2'3'-13C1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar Hsd/Cpb: WU
- Source: Harlan & Winkelmann GmbH, Gartenstraße 27, 33178 Borchen, Germany
- Age at study initiation: 7 weeks at the time of delivery
- Weight at study initiation: a mean weight of about 200 g: 194 - 205 g at the time of administration; 206 - 215 g at the time of sacrifice
- Fasting period before study: yes, last feeding ca. 16 hours prior to dosing, next feeding ca. 6 hours after dosing
- Housing: Conventional hygienic conditions in air-conditioned rooms
- Individual metabolism cages: yes After administration of the radiolabelled test item, the rats were kept individually in Makrolon® metabolism cages. With these cages, an almost quantitative and separate collection of urine and faeces was possible.
- Diet (e.g. ad libitum): ad libitum (Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland (ca. 16 g per animal and day),
- Water (e.g. ad libitum): Tap water from the local mains supply (ad libitum)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 53 - 60
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The [14C]labelled test item (BECH 2014; specific radioactivity: 4.31 MBq/mg) was shipped as aqueous solution (54.86 mg IDS, Na4 salt in 15 g water). Before preparation of the administration solution, the identity was confirmed by LC-MS/MS. The radiochemical purity (sum of isomer 1 and 2) was checked by radio-HPLC. The [13C]labelled test item was shipped as solid material (1027 mg) and was dissolved in 10 g water. The unlabelled test item was shipped as aqueous solution (34.7% = 347 mg IDS, Na4 salt/g solution).
The preparation of the administration solution used for the ADME-experiment (test 1) is shown in the table 1.
Based on the calculation presented in the table 1 a final specific radioactivity of 0.039 MBq/mg (1.06 u.Ci/mg) was obtained. The administration solution was prepared one day before dosing. The radiochemical purity and concentration was determined by radio-HPLC and by LSC, respectively. The calibration results of the administration solution served as a reference for the calculation of the total radioactivity in the biological samples.

For test 2, 3.47 g of the unlabelled test items (34.7%) solution was filled up with water to a total weight of 12 g.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:

Duration and frequency of treatment / exposure:

single administration

Doses / concentrations

Remarks:

Doses / Concentrations:
Test 1: 1000 mg/kg bw (nominal);
Test 2: 1000 mg/kg bw (nominal).

No. of animals per sex per dose / concentration:

5 animals dosed with the radiolabelled test item (test 1)
5 animals dosed with the non-radiolabelled test item (test 2)

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

None.

Details on study design:

- Dose selection rationale: Five male rats each were assigned to their respective test groups 1 and 2 as noted in Table 2. They received the calculated volume by oral gavage using a syringe attached to an animal-feeding knob cannula. The volume was based on the nominal average animal weight of 200 g. The concentration of each administration solution was calculated to reach an administered amount of about the nominal value of the parent compound per kg body weight (bw). Due to different animal weights at administration, the actual dose per kg varied slightly with the body weight. Two millilitre of the respective administration solution were dosed to each animal. The amounts of the test compound administered in the different experiments are shown in the Table 3.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, liver, kidney, GIT, skin and carcass
- Time and frequency of sampling: see Table 2

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling: Urine (0 - 4 h, 4 - 8 h, 8 - 24 h, 24 - 48 h and 48 - 72 h); faeces (0-24 h); see also Table 2
- From how many animals: all animals (pooled)
- Method type(s) for identification (HPLC-UV, Liquid scintillation counting, NMR)

- Limits of detection and quantification: For all samples, the limit of detection (LOD) was predefined on the base of the background radioactivity counting rate which was in the range between 12 - 29 cpm (in average 21 cpm), depending on the instrument used. The LOD was established as twice the average background radioactivity. Single background subtraction and a quench and counting efficiency correction for transformation of gross counts (cpm) into net counts (dpm) were automatically performed by the instruments. Therefore, samples with individually measured values below 21 dpm (after correction for the background radioactivity) were not quantified and labelled as < LOD or LOQ in the respective tables. The limit of quantification (LOQ) was set on the level of LOD.
A typical calculation example for the LOD of combustion/LSC is given below:
Limit of detection = (BKG x Df)/representative sample weight x (1/SA) = (21dpm x 0.2/ 0.05 g) x (1 µg/2363 dpm) = 0.036 mg/kg
where:
BKG : average background radioactivity (21 dpm);
Df : freeze drying factor (e.g.: 0.2), (= weight of sample after lyophilisation / weight of sample prior to lyophilisation); this factor is equal to 1 in case of liquid samples;
0.05 : typical amount of sample used for radioactive measurement [g];
SA: specific radioactivity of the test item [dpm/ug] = 2363 dpm/µg.
The counting times of the samples were generally between seconds and 20 minutes depending on the amount of radioactivity present in the sample. The measurement was stopped after reaching a 2-a error of 0.7%. If this error was not reached within 20 minutes, the measurement was stopped and the 2-a error of the dpm-value (LKB/Wallac 1219 Spectral) or cpm-value (Beckman counter) reached at this time was recorded.

OTHER: Determination of Osmolality, pH and Metals in Urine and Plasma
The osmolality, pH-values and amount of selected metals in urine and plasma samples from rats which received nearly the same amount of the unlabelled IDS, Na4 salt (test 2) were additionally determined and compared to control samples obtained from untreated rats.

Statistics:

see attached background material.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

at least 37 % of the administered dose becomes systemically available.

Main ADME results 2

Type:

distribution

Results:

2.2 % were found in the total body. The relatively highest equivalent concentrations were detected in the liver (50 mg/kg), followed by kidney (36 mg/kg) and skin (25 mg/kg). For erythrocytes, plasma, carcass and GIT, the values were below 20 mg/kg.

Main ADME results 3

Type:

metabolism

Results:

The unchanged substance was recovered in the urine (99.5 % during 0-72 h) and in the faeces (93.3 % during 0-24 h). A biotransformation pathway was not proposed because no single unknown metabolite was higher than 0.2 % in urine and 2 % in faeces.

Main ADME results 4

Type:

excretion

Results:

The excretion of radioactivity via urine and faeces was fast and almost completed at 24 h after administration. The by far major part of the dose (68.7 %) was excreted with faeces and 34.7 % with urine.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The absorption of the test item related radioactivity from the gastrointestinal tract and distribution into the different organs and tissues was investigated by determination of the test item equivalent concentrations (= TRR) in plasma at fixed dates over the whole testing period. The individual values for the equivalent and dose-normalised concentrations are given in Table 4. Oral administration of [14C]IDS, Na4 salt to male rats resulted in a rapid appearance of substantial plasma concentrations of 14C at the first time point of 10 min postdose and quantifiable concentrations at all time points thereafter. The time course of the plasma concentrations showed a biphasic behaviour (Figure 1). A first portion of the dose of was rapidly absorbed from the gastrointestinal tract immediately after dosing without a recognisable lag-time and reached a maximum concentration (Cmax) of 186 mg/kg after 20 min. This value corresponded to 19.4% of the equidistribution concentration of the administered dose. Until 4 hours after dosing, the concentration dropped to about 10 mg/kg which indicated a fast elimination of the test item related radioactivity from the central compartment (blood) towards the peripheral compartments (organs and tissues; 1st elimination phase). A second increase of the TRR-values to approx. 65 mg/kg until 24 hours was measured thereafter followed by a decrease to a final value of 17 mg/kg of 72 hours post dosing (2nd elimination phase). An apparent saturation of absorption due to the high administered amount during the first phase might have been the reason for this biphasic behaviour. An exact value for the absorption rate could not be taken from these figures. But taking the urinary excretion behaviour and the remaining radioactivity in the body at sacrifice into account it can be concluded that at least 37% of the administered dose becomes systemically available.

A summary of the key pharmacokinetic parameters of total [14C] in plasma is presented in Table 5.The half-life for the first elimination phase (t1/2e1) which lasted from 40 min to 4 h postdose amounted to 0.7 h. For the second elimination phase from 24 to 72 h postdose, the terminal half-life (t1/2 e2) and the terminal elimination rate accounted for 25 h and 0.028 [h-1], respectively. The mean area under the curve from 0.16 - 4 h (AUC 0.16 -4) was 296 mg x h/kg (9%), from 4 - 72 h (AUC 4 - 72) 2393 mg x h/kg (72%), and the mean AUC extrapolated from 72 h to infinity (AUC 72 - ∞)was 602 mg x h/kg (18%). As shown for the second AUC-value from 4 - 72 h postdose, a similar high value was calculated for the respective mean area under the data value (AUD 4 -72) of 2393 mg x h/kg. The second absorption and elimination phases contributed obviously mainly to the systemic exposure of the test item and/or its metabolites. The mean residence time (MRT 0 - ∞) of 45 h was relatively high but in view of a low accumulation factor of 1.4 a retention of test item related radioactivity in the body could be excluded.

Details on distribution in tissues:

The total radioactive residues in the organs and tissues (expressed as equivalent and dose normalised concentrations as well as percentage of administered dose) were determined at sacrifice (Table 8). In relation to the administered dose, only 2.2 % were found in the total body. The carcass, skin and liver contributed in decreasing order mostly to this value. The relatively highest equivalent concentrations were detected in the liver (50 mg/kg), followed by kidney (36 mg/kg) and skin (25 mg/kg). For all other samples (erythrocytes, plasma, carcass and GIT), the values were below 20
mg/kg.

Details on excretion:

The excretion of radioactivity via urine and faeces is reported in Table 7. In contrast to the high administered amount, the excretion was fast and almost completed at 24 h after administration. The by far major part of the dose (68.7%) was excreted with faeces and 34.7% with urine.

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#1

Toxicokinetic parameters:

Cmax: 186 mg/kg

Toxicokinetic parameters 2

Test no.:

#1

Toxicokinetic parameters:

Tmax: 0.33 h

Toxicokinetic parameters 3

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 0.73 h

Toxicokinetic parameters 4

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 25.14 h

Toxicokinetic parameters 5

Test no.:

#1

Toxicokinetic parameters:

AUC: 296.38 mg x h/kg (for 0.16-4 h)

Toxicokinetic parameters 6

Test no.:

#1

Toxicokinetic parameters:

AUC: 23.92.71 mg x h/kg (for 4-72 h)

Toxicokinetic parameters 7

Test no.:

#1

Toxicokinetic parameters:

AUC: 602.4 mg x h/kg (for 72- ∞ h)

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

Urine pool samples from the different time period were directly taken for elucidation of the parent compound and metabolites. The 0 - 24 h faeces pool sample was conventionally extracted with ACN/water mixtures. The extraction efficiency was 97 % proving the adequacy of this procedure. The combined extracts were directly used for analysis. Faeces samples from the collection periods 24 - 48 h and 48 - 72 h were not analysed because only 3 % of the faecal radioactivity was found in these samples (Table 7 of IUCLID dataset). The parent compound only was unambiguously identified in the urine and faeces samples.
The identification rate for the unchanged IDS, Na4 salt was high and amounted 99.5 % for urine (0 - 72 h; Table 9 of IUCLID dataset) and 93.3 % for faeces (0 - 24 h; Table 10 of IUCLID dataset). Relating to the administered dose, IDS, Na4 salt amounted to 34.5 % in urine and 61.2 % in faeces (Table 11 of IUCLID dataset). No single unknown metabolite was higher than 0.2 % in urine and 2 % in faeces.
Because the unchanged IDS, Na4 salt was the only identified compound and significant degradation did not occur a biotransformation pathway was not proposed.

Any other information on results incl. tables

Table 4 . Test 1 : Time courses of the equivalent and dose normalized concentrations in plasma of male rats after administration of 1000 mg [1 4C]IDS, Na4 salt/kg bw

Equivalent concentration С [mg/kg]
Time	Animal no.
(h p. admin.)	347	348	349	350	351	Mean	CV
0.16	125.0	82.66	153.0	102.0	189.0	130.0	32.2
0.33	178.0	138.0	182.0	192.0	239.0	186.0	19.5
0.66	185.0	142.0	163.0	198.0	221.0	182.0	16.8
1.0	172.0	130.0	134.0	169.0	212.0	163.0	20.4
15	108.0	84.57	88.55	114.0	135.0	106.0	19.2
2.0	56.55	36.3	42.63	45.92	52.08	46.7	17.0
3.0	24.96	13.68	12.83	13.05	16.90	16.28	31.4
4.0	11.50	9.15	9.53	8.50	12.27	10.19	15.8
6.0	8.15	16.41	7.65	6.65	52.11	18.19	106.4
8.0	12.19	47.16	9.03	7.63	12.71	17.74	93.5
24.0	63.36	102.00	57.69	54.51	44.85	64.51	34.2
32.0	38.00	85.29	44.29	50.04	39.42	51.41	38.0
48.0	19.13	48.65	25.21	26.96	23.26	28.64	40.4
56.0	17.64	45.13	21.80	24.17	21.34	26.02	42.1
72.0	11.67	29.70	15.30	16.34	14.21	17.44	40.5
Dose normalized concentration CN
Time	Animal no.
(h p. admin.)	347	348	349	350	351	Mean	CV
0.16	0.131	0.086	0.158	0.108	0.198	0.136	32.3
0.33	0.186	0.143	0.189	0.202	0.250	0.194	19.9
0.66	0.193	0.147	0.169	0.209	0.231	0.190	17.3
1.0	0.180	0.135	0.139	0.177	0.222	0.170	20.9
1.5	0.113	0.088	0.092	0.120	0.141	0.111	19.7
2.0	0.059	0.038	0.044	0.048	0.055	0.049	17.4
3.0	0.026	0.014	0.013	0.014	0.018	0.017	31.7
4.0	0.012	0.009	0.010	0.009	0.013	0.011	16.0
6.0	0.009	0.017	0.008	0.007	0.055	0.019	106.7
8.0	0.013	0.049	0.009	0.008	0.013	0.018	92.9
24.0	0.066	0.106	0.060	0.057	0.047	0.067	33.7
32.0	0.040	0.088	0.046	0.053	0.041	0.054	37.5
48.0	0.020	0.050	0.026	0.028	0.024	0.030	39.8
56.0	0.018	0.047	0.023	0.025	0.022	0.027	41.5
72.0	0.012	0.031	0.016	0.017	0.015	0.018	40.0

Table 5. Test 1 : Pharmacokinetic data analysis of the mean equivalent concentrations in plasma of male rats after administration of 1000 mg [14C]IDS, Na4 salt/kg bw

Pharmacokinetic non-compartmental data analysis
Time	Measured	Calculated	Pharmacokinetic parameters
(h p. admin.)	value [mg/kg]	value [mg/kg]
0.16	130.0	—	Сmах[mg/kg]	186.0
0.33	186.0	—	tmax[h]	0.33
0.66	182.0 *)	—	t1/2 e1[h]	0.73
1.0	163.0 *)	—	t1/2 e2 = terminal half-life) [h]	25.14
1.5	106.0 *)	—	Terminal elimination rate [1/hour] = к	0.028
2.0	46.7 *)	—	Intercept at time 0 [mg/kg] = С (0)	120.9
3.0	16.28 *)	—	Correlation coefficient	0.9841
4.0	10.19 *)	—	AUD(0.16-4)[mg x hour/kg]	296.38
6.0	18.19	—	AUD(4-24)[mg x hour/kg]	722.31
8.0	17.74	—	AUD(24-72)[mg x hour/kg]	1670.40
24.0	64.51 **)	62.4^+)	AUC(0.16-4)[mg x hour/kg]	296.38
32.0	51.41 **)	50.0^+)	AUC(0.16-4)[%]	8.98
48.0	28.64 **)	32.2^+)	AUD(4-72)[mg x hour/kg]	2392.71
56.0	26.02 **)	25.8^+)	AUD(4-72)[%]	72.46
72.0	17.44 **)	16.6^+)	AUD(72 -∞)[mg x hour/kg]^++	602.40
			AUD(72 ∞-)[%]^++	18.24
			MRT (0-∞)[hour]	45.26
			Accumulation factor (AF)	1.36

Table 6. Test 1 : Balance of radioactivity in excreta as well as organs and tissues of male rats after administration of 1000 mg [1 4C]IDS, Na4 salt/kg bw

Radioactivity in percent of dose administered
	Animal no.
	347	348	349	350	351	Mean	CV
Urine	43.20	18.27	25.32	28.38	58.18	34.67	46.1
Faeces	59.86	75.95	83.93	82.21	41.30	68.65	26.2
Total excreted	103.06	94.22	109.25	110.59	99.48	103.32	5.9
Skin	0.38	1.01	0.63	0.65	0.61	0.66	34.7
Sum Organs	0.91	2.02	1.39	1.40	1.21	1.38	29.3
Body without GIT	1.29	3.03	2.01	2.04	1.82	2.04	31.0
GIT	0.15	0.31	0.16	0.19	0.16	0.20	32.6
Total in body	1.44	3.34	2.18	2.24	1.98	2.23	31.0
Balance	104.50	97.55	111.40	112.80	101.50	105.60	6.2
Radioactivity in percent of dose recovered
	Animal no.
	347	348	349	350	351	Mean	CV
Urine	41.34	18.73	22.73	25.15	57.34	33.06	48.6
Faeces	57.28	77.85	75.32	72.86	40.71	64.80	24.2
Total excreted	98.62	96.58	98.05	98.01	98.05	97.86	0.7
Skin	0.36	1.04	0.56	0.57	0.60	0.63	39.5
Sum Organs	0.87	2.07	1.24	1.24	1.19	1.32	33.7
Body without GIT	1.23	3.11	1.81	1.81	1.79	1.95	35.5
GIT	0.15	0.31	0.15	0.17	0.16	0.19	38.0
Total in body	1.38	3.42	1.96	1.98	1.95	2.14	35.6
Norm.-factor	0.96	1.03	0.90	0.89	0.99	0.95	6.2

Osmolality

The osmolality of the 4 hour urine samples from the treated rats were by a factor of 6.2 lower (mean of 143 mOsmol/kg) than the control urine sample from untreated rats (884 mOsmol/kg). After about 8 hours, the values increased clearly together with the total urine volume and entered into a plateau phase at about 24 h postdose. The highest value measured at 48 hours after administration (568 mOsmol/kg). The curve progression was similar to the urinary excretion curve which showed that an assumed osmotic imbalance was recovered within this period probably due to the final excretion of the unchanged test item. The plasma value of 314 mOsmol/kg at sacrifice showed no discrepancy to a respective value obtained from untreated rats (302 mOsmol/kg).

pH-Values and Metals

An only slight decrease of the pH-values of the urine samples from treated rats (pH 6.9 - 7.8) compared to a reference-value from untreated rats (pH 8.4) was measured.

The time courses of inorganic metals however showed clear differences. Sodium was reduced by nearly the half compared to the reference-value until 4 hours postdose. It increased significantly until 24 h postdose to a peak level of about 1700 mg/L before it declined to 686 mg/L at sacrifice. The curve progression was comparable to the plasma curve of the test item related radioactivity. After low levels at 4 hours, a steady increase until test end was determined for calcium and also for magnesium until 48 h postdose. The final values for these two metals were higher than the respective reference-values. The 4 to 24 hours values for iron and zinc were higher than the reference values. They declined afterwards until test end. The final values were comparable to the reference values for zinc and higher for iron. For the respective plasma data from the treated and untreated rats, slight differences were recognized for the metals only. Compared to the reference values, those for calcium and iron were by a factor of 1.3 higher and those for sodium and zinc by a factor of 1.2 lower. Equal amounts were measured for magnesium.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The kinetic and metabolic behaviour of [14C]Iminodisuccinate, Na4 salt in male rats can be characterised by the following observations:
• A first portion of the administered radioactivity was rapidly absorbed, widely distributed into organs and tissues and rapidly eliminated from the body via urine and faeces.
• For a second portion, the absorption and elimination periods were significant longer than for the first one which indicated a higher systemic exposure to the test item related radioactivity during this phase.
• The distribution of the radioactivity within the central compartments of the body (i.e. blood, liver, and kidney) showed a distinctive preference towards the liver and - to a lower extent - to the kidney which had the highest TRR (total radioactive residue) values at sacrifice.
• IDS, Na4 salt was metabolically stable as no metabolites were detected in the excreta in significant amounts.
• From the renal and faecal excretion rate as well as the low accumulation factor it was concluded that the residual radioactivity in the organs and tissues of the rat are subject to further elimination.
• The administration of IDS, Na4 led to transient changes of the urinary osmolality-and pH-values within the test period of 3 days. Based on a high variability of urine pH- and osmolality-values in animal species used in toxicological studies, these effects however were regarded as insignificant. Differences were obtained in the amount of metals in urine and plasma samples between treated and untreated rats.
Based on these results, it is believed that the depletion of residues and metabolic behaviour of [14C]Iminodisuccinate, Na4 salt in male rats is sufficiently understood.

Executive summary:

The present study describes investigations on the pharmacokinetic behaviour (absorption, distribution, excretion) and metabolism of the complexing agent IDHA for metal ions. The test material was labelled with 14C and 13C in the 2 and 3 as well as 2' and 3' - positions of imminodisuccinic acid. The radiolabelled and unlabelled test material was administered to two groups of 5 male Wistar rats, respectively, at dose level of 1000 mg/kg bw. The rats received the test item by oral gavage as an aqueous solution. They were sacrificed 72 h after dosing. The total radioactivity that included the test item and possible metabolites was determined in plasma samples, the excreta (urine and faeces) as well as in organs and tissues. The metabolism was investigated by radio-HPLC and spectroscopic methods in selected urine samples and faeces extract. Further, osmolality in urine as well as pH-values and metals in urine and plasma samples collected from rats which received the unlabelled test item were investigated.

Absorption and pharmacokinetic behaviour

The absorption of the test item related radioactivity from the gastrointestinal tract and distribution into the different organs and tissues was investigated by determination of the test item equivalent concentrations (= TRR) in plasma at fixed dates over the whole testing period.

Oral administration of [14C]IDS, Na4 salt to male rats resulted in a rapid appearance of substantial plasma concentrations of 14C at the first time point of 10 min postdose and quantifiable concentrations at all time points thereafter. The time course of the plasma concentrations showed a biphasic behaviour. A first portion of the dose of was rapidly absorbed from the gastrointestinal tract immediately after dosing without a recognisable lag-time and reached a maximum concentration (Cmax) of 186 mg/kg after 20 min. This value corresponded to 19.4% of the equidistribution concentration of the administered dose. Until 4 hours after dosing, the concentration dropped to about 10 mg/kg which indicated a fast elimination of the test item related radioactivity from the central compart¬ment (blood) towards the peripheral compartments (organs and tissues; 1st elimination phase). A second increase of the TRR-values to approx. 65 mg/kg until 24 hours was measured thereafter followed by a decrease to a final value of 17 mg/kg of 72 hours post dosing (2nd elimination phase). An apparent saturation of absorption due to the high administered amount during the first phase might have been the reason for this biphasic behaviour. An exact value for the absorption rate could not be taken from these figures. But taking the urinary excretion behaviour and the remaining radioactivity in the body at sacrifice into account it can be concluded that at least 37% of the administered dose becomes systemically available.

The half-life for the first elimination phase (t1/2 e1) which lasted from 40 min to 4 h postdose amounted to 0.7 h. For the second elimination phase from 24 to 72 h postdose, the terminal half-life (t1/2 e2) and the terminal elimination rate accounted for 25 h and 0.028 [h-1], respectively. The mean area under the curve from 0.16 - 4 h (AUC 0.16 - 4) was 296 mg x h/kg (9%), from 4 - 72 h (AUC 4 - 72) 2393 mg x h/kg (72%), and the mean AUC extrapolated from 72 h to infinity (AUC 72 - ∞ was 602 mg x h/kg (18%). As shown for the second AUC-value from 4 - 72 h postdose, a similar high value was calculated for the respective mean area under the data value (AUD 4 - 72) of 2393 mg x h/kg. The second absorption and elimination phases contributed obviously mainly to the systemic exposure of the test item and/or its metabolites. The mean residence time (MRT 0 - ∞) of 45 h was relatively high but in view of a low accumulation factor of 1.4 a retention of test item related radioactivity in the body could be excluded.

Excretion

The excretion of radioactivity via urine and faeces was fast and almost completed at 24 h after administration. The by far major part of the dose (68.7%) was excreted with faeces and 34.7% with urine.

Radioactive residues in organs and tissues at sacrifice

The total radioactive residues in the organs and tissues were determined at sacrifice. In relation to the administered dose, only 2.2% were found in the total body. The carcass, skin and liver contributed in decreasing order mostly to this value. The relatively highest equivalent concentrations were detected in the liver (50 mg/kg), followed by kidney (36 mg/kg) and skin (25 mg/kg). For all other samples (erythrocytes, plasma, carcass and GIT), the values were below 20 mg/kg. Because of the high the renal and faecal excretion rate it is expected that the small amounts of residual radioactivity in the organs and tissues of the rat are subject to further elimination.

Metabolism

The metabolism of [14C]IDS, Na4 salt was investigated in selected urine and faeces samples. The radio-HPLC-profiles were very similar and highly comparable to each other. Significant qualitative differences did not occur. The identification rate for the unchanged IDS, Na4 salt was high and amounted 99.5% for urine (0 - 72 h) and 93.3% for faeces (0 - 24 h). Relating to the administered dose IDS, Na4 salt amounted to 34.5% in urine and 61.2% in faeces. A biotransformation pathway was not proposed because no single unknown metabolite was higher than 0.2% in urine and 2% in faeces.

Determination of osmolality, pH and metals in urine and plasma samples

The osmolality, pH-values and amount of selected metals in urine and plasma samples from rats which received nearly the same amount of the unlabelled IDS, Na4 salt (test 2) were additionally determined.

The administration of IDS, Na4 led to transient changes of the urinary osmolality- and pH-values within the test period of 3 days. Based on a high variability of urine pH- and osmolality-values in animal species used in toxicological studies [1], these effects however were regarded as insignificant.

The osmolality of the 4 hour urine samples from the treated rats were by a factor of 6.2 lower (mean of 143 mOsmol/kg) than the control urine sample from untreated rats (884 mOsmol/kg). After about 8 hours, the values increased clearly together with the total urine volume and entered into a plateau phase at about 24 h postdose. The highest value measured at 48 hours after administration (568 mOsmol/kg). The curve progression was similar to the urinary excretion curve which showed that an assumed osmotic imbalance was recovered within this period probably due to the final excretion of the unchanged test item. The plasma value of 314 mOsmol/kg at sacrifice showed no discrepancy to a respective value obtained from untreated rats (302 mOsmol/kg).

The time courses of metals in urine samples showed clear differences. Sodium was reduced by nearly the half compared to the reference-value until 4 hours postdose. It increased significantly until 24 h postdose to a peak level of about 1700 mg/L before it declined to 686 mg/L at sacrifice. The curve progression was comparable to the plasma curve of the test item related radioactivity. After low levels at 4 hours, a steady increase until test end was determined for calcium and also for magnesium until 48 h postdose. The final values for these two metals were higher than the respective reference-values. The 4 to 24 hours values for iron and zinc were higher than the reference values. They declined afterwards until test end. The final values were comparable to the reference values for zinc and higher for iron.

For the respective plasma data from the treated and untreated rats, slight differences were recognized for the metals likewise. Compared to the reference values, those for calcium and iron were by a factor of 1.3 higher and those for sodium and zinc by a factor of 1.2 lower. Equal amounts were measured for magnesium.