SUMILIZER GP

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:

05 Dec 2007 - 28 Jul 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

mass balance

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted in 1984

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted in 2010

Deviations:

yes

Remarks:

age of the animals not reported

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland

Radiolabelling:

yes

Species:

rat

Strain:

other: CD (SD) IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kissleg, Germany
- Weight at study initiation: 238 ± 21 g (males) and 217 ± 21 g (femlaes)
- Housing: In groups of 1-3/sex under conventional hygienic conditions in Makrolon cages with standard soft wood bedding during acclimation. Individually in metabolism cages during the course of the study.
- Diet: Pelleted 3433 Kliba rat maintenance diet (Provimi Kliba AG, Kaiseraugstm Switzerland), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days
- Health status: The health status of the treated animals was checked visually at daily intervals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: aqueous methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Stock solutions of 14C-labeled test items were prepared by mixing aliquot amounts of unlabeled test item with 14C-labeled test item (MOH or TBPH-label) in 10 mL of hexane (10 mg/kg bw, groups 3, 4, 7, and 8) or dichloromethane (1000 mg/kg bw, groups 5 and 6) in a volumetric flask.

MOH-labelled test item, 10 mg/kg bw: (groups 3+4)
9.95 mL of the stock solution were transferred into a volumetric flask and the solvent was evaporated under a nitrogen stream. The test item was pre-dissolved in 1.5 mL of DMSO and 0.5% methocel solution was added to a total volume of 30 mL under stirring. Test item concentration and homogeneity was verified by LSC measurements of triplicate samples. The resulting test item concentration in the application solution was 0.98 mg/g.

TBPH-labelled test item, 10 mg/kg bw: (groups 7+8)
9.85 mL of the stock solution were transferred into a volumetric flask and the solvent was evaporated under a nitrogen stream. The test item was pre-dissolved in 1.5 mL of DMSO and 0.5% methocel solution was added to a total volume of 30 mL under stirring. Test item concentration and homogeneity was verified by LSC measurements of triplicate samples. The resulting test item concentration in the application solution was 0.98 mg/g

TBPH-labelled test item, 1000 mg/kg bw: (groups 5+6)
The complete stock solution was transferred into a 100 mL round bottom flask and 30 mL 0.5% methocel solution were added. The dichloromethane solvent was evaporated in a nitrogen stream under vigorous stirring and further 10-12 mL methocel solution added to replace loss by evaporation and to reach a suitable consistency for administration. The concentration of the test item solution was determined by LSC to be 67.8 mg/g. The formulation was kept under permanent stirring until administration.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

equivalent to 9.80 ± 0.18 mg/kg bw in males and 9.98 ± 0.30 mg/kg bw in females;
low dose MOH-label, groups 3 (males) and 4 (females), corresponding to 25 MBq (0.67 mCi)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

equivalent to 9.65 ± 0.19 mg/kg bw in males and 10.10 ± 0.30 mg/kg bw in females;low dose TBPH-label, groups 7 (males) and 8 (females), corresponding to 25 MBq (0.67 mCi)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 992 ± 45 mg/kg bw in males and 991 ± 73 mg/kg bw in females;
high dose TBPH-label, groups 5 (males) and 6 (females), corresponding to 50 MBq (1.35 mCi)

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on study design:

- Dose selection rationale: Comparison of the excretion balance after administration of 2 labels (MOH and TBPH) and between a high dose (1000 mg/kg bw) and a low dose (10 mg/kg bw).

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, cage washes, blood, carcass and tissues. As tissues, the adrenal glands, blood, brain, epididymes , eyes , fat, femur, heart, kidney, large intestinal tract, liver, lung , muscle , ovaries, pancreas , pituitary gland, plasma , prostate gland , sciatic nerve, skin (back region), small intestinal tract, spinal cord, spleen, stomach, testes , thymus, thyroid gland, urinary bladder and uterus were collected. Additionally, the contents of the stomach, large intestine and small intestine of each animal were pooled and analysed.
- Time and frequency of sampling:
urine: 0-8, 8-24, 24-48, 48-72, 72-96, 96-120, 120-144, and 144-168 h after administration
faeces: 0-24, 24-48, 48-72, 72-96, 96-120, 120-144, and 144-168 h after administration
blood: at sacrifice after 168 h
organs/tissues/carcass: at sacrifice after 168 h
cage wash: 0-8 and 0-24 h after administration

SAMPLE PROCESSING
CAGE WASH: At the end of the each interval 0-8 and 0-24 h collection intervals, the metabolism cages were rinsed with a small amount of purified water (about 5-10 mL) to remove any remaining droplets of urine. The rinsings were added to the initially sampled urine and the radioactivity was evaluated. At the end of the study, the cages were washed with ethanol, water/acetone (50/50, v/v) and/or appropriate solvnts for the determination of any residual radioactivity.
URINE: Subsamples of urine were mixed at room temperature with liquid scintillation counting (LSC) cocktail (IRGA-SAFE PLUSTM, Perkin Elmer) analysed by LSC. Counting was performed using a Packard TRI-CARBTM Liquid Scintillation Analyser (model 2500 TR).
FAECES: Faeces samples were homogenised wet by addition of water (about 1+2, w/v). After homogenization, the radioactivity was determined from appropriate aliquots after solubilisation with tissue solubiliser (Solvable, Perkin Elmer).
CARCASS: The carcass was homogenized and the radioactivity was determined from appropriate aliquots of the homogenate after solubilisation with tissue solubiliser (Solvable, Perkin Elmer).
ORGANS / BLOOD: Subsamples were digested with tissue solubiliser (Solvable, Perkin Elmer), followed by determination of radioactivity.
INTESTINAL TRACT: The contents of the stomach, large intestine and small intestine of each animal were pooled, homogenised wet by addition of water (about 1+2, w/v and radioactivity was determined from appropriate aliquots after solubilisation with tissue solubiliser (Solvable, Perkin Elmer).
PLASMA: Plasma samples were directly measured for radioactivity.
BONE: The femur was combusted and radioactivity was determined thereafter.

EXTRACTION OF FAECES POOLS
Faeces samples of the 24 and 48 h collection time points were pooled per group and extracted 3 times with neural acetonitrile, followed by a further extraction with 0.1 M HCl/Acetonitrile (2:8). The 0-24 h faeces pools of the group 3 animals were extracted with ethyl acetate before the acidic acetonitrile extraction. As this additional step did not improve the recovery, all other pools were processed without ethyl acetate extraction. The extracts were combined, concentrated and analysed by HPLC.

PURIFICATION OF URINE POOLS
Four pools were generated of the individual urine samples of the group 3 and 4 animals, collection intervals 0-8 and 8-24 h with 2 mL urine per animal and collection interval. A Separtis Isolute RP18 solid phase extraction column1 (5 g sorbent mass) was pre-conditioned with 0.1% aqueous ammonium acetate. 8 mL of urine per group were transferred onto the column and eluted with 0.1% aqueous ammonium acetate (approximately 11 mL per pool) and 100% methanol (4.1 - 4.4 mL). Total activity in all fractions was determined, the total recovery was 88- 102%. The methanol fractions were analysed by HPLC.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling:
urine: 0-8, 8-24 and 24-48 h after administration
faeces: 0-24 and 24-48 h after administration
- From how many animals: sampled were pooled for each sex and dose group
- Method types for identification: HPLC with UV detector and radiodetector, Liquid scintillation counting (LSC)
- Limits of detection and quantification: Limits of detection (LOD) and limits of quantification (LOQ) for liquid scintillation counting were calculated according to L.A. Currie (Anal. Chem. 40, 586 (1968)) from the specific radioactivity of the test item.

ANALYTICAL METHODS :

Measurement of radioactivity
Radioactivity in all specimens was determined on Packard liquid scintillation counters (e.g. TRICARB 2500 TR, 2550 TR/LL or 2900 TR) using the Transformed Spectral Index of the External Standard Spectrum (tSIE) method for quench correction. All measurements were performed as far as possible at least in duplicate for a counting time allowing a counting error below 1% or for a maximal counting time of 10 minutes. All measurements were performed with scintillation background correction. In case duplicates differed by more than 10% from the mean value, as far as possible re-analyses were performed using fresh aliquots (except for measurements below 100 dpm).

The following scintillation mixtures were used:
A: Irga-Safe Plus (Perkin Elmer)
B: Solvable (Perkin Elmer)
Water: Milli-Q water (Milli-Q plus 185, Millipore)

HPLC analysis
HPLC analysis was used for the determination of the purity of the administration solutions and for the determination of metabolites in urine and feces. The retention time of parent compound was determined for comparison with chromatograms of faeces samples. Analyses were performed with a HPLC-system consisting of a gradient pump (Merck-Hitachi L-6200), an autosampler (Merck-Hitachi AS-2000 A), an UV-detector (Merck-Hitachi L-4000), a radiodetector and a data processing system (Packard FLOW-ONE, Beta A 500).
For details on the methods used, please refer to Table 1 under "Any other information on materials and methods incl. tables".

Statistics:

The concentration of radioactivity in blood and plasma were expressed as parent equivalents on a fresh weight basis (pg eq/mL or g), and compared with the limits of quantification. Limits of detection (LOD) and limits of quantification (LOQ) for liquid scintillation counting were calculated according to L.A. Currie (Anal. Chem. 40, 586 (1968)) from the specific radioactivity of the test item.

Type:

absorption

Results:

Both the MOH and the TBPH label were completely recovered in excreta, organs, tissues and carcass and in the cage wash. The mass balance in all groups ranged between 91.7-101%.

Type:

distribution

Results:

Residues in the tissues at sacrifice were generally low. With the MOH label, the highest concentrations were found in the liver & kidney. Residues of the TBPH label were primarily found in adrenal glands, thyroid gland, liver, fatty tissue & ovaries.

Type:

excretion

Results:

Both labels of the test item were rapidly excreted. 91-100% of the applied dose was recovered in urine and faeces within the first 48 h after administration. The major route of excretion was via the faeces.

Type:

metabolism

Results:

15 metabolites detected in faeces (5 attr to unchanged parent/references, 10 unknown). 10 mg/kg bw (MOH/TBPH): the major metabolite was attr to ref.compound NP-2018 (45-71%). 1000 mg/kg bw: the major metabolite was attr to unchanged parent (62-65%).

Details on absorption:

The mass balance in all test groups ranged from 91.7 - 101% (refer to Table 2 under "Any other information on results incl. tables"). As the main excretion route was via the faeces (>90% of the dose for all test groups), the extent of oral absorption could not be determined.

Details on distribution in tissues:

In males and females of both labels and in all dose groups, the major part of the administered dose was excreted via the faeces. At the time of sacrifice, 0.065 - 0.27% (males) and 0.03 – 0.195% (females) of the dose were found in the organs, tissues, carcass and the content of the gastrointestinal tract. After administration of 10 mg/kg bw MOH-labelled test item, the highest concentrations found were in the kidneys and in the liver.
After administration of 10 or 1000 mg/kg bw TBPH-labelled test item, the highest amounts were found in the adrenal glands, liver, thyroids, ovaries and in fat. There were no marked differences found between males and females and between the low and the high dose group. For details, please refer to Table 3 under "Any other information on results incl. tables".

Details on excretion:

For both labels and both dose groups, the major excretion route was via the faeces (90.5 - 101% of the dose). There was no difference between males and females. Urinary excretion accounted for 1.14 – 1.77% of the dose for the MOH label and 0.05% or less for the TBPH label. More than 90% of the dose was excreted within 48 h post dosing. For details, please refer to Table 2 under "Any other information on results incl. tables".

Metabolites identified:

yes

Details on metabolites:

Metabolites were identified in the faeces (all treatment groups) and in the urine (MOH-labelled treatment group only).
In the faeces, 87 – 115% of the administered dose were recovered (refer to Table 4 under “Any other information on results incl. tables”). A total of 15 metabolite fractions were identified, 10 of them with unknown origin. Within the low dose groups, about 3.6 – 6.3% (groups 3 and 4 with 10 mg/kg bw MOH-labelled substance) and 17-23% (groups 7 and 8 with 10 mg/kg bw TBPH-labelled substance) of the parent compound were identified in the faeces. The main metabolite in the low dose group of both labels was F4, attributed to reference substance NP-2018.
In the high dose group (groups 5 and 6 with 1000 mg/kg bw TBPH-labelled substance), 62-65% of the radioactivity were attributed to unchanged parent substance and the main metabolite F4 accounted for 12-14% of the dose. The MOH label (groups 3 and 4) formed a more complex metabolite pattern as compared to the TBPH label. The relatively polar fractions F10-F15 were only observed with the MOH label, only F11 (2.4 – 3.6%), attributed to MOH, was more abundant than 1% of the dose. For details, refer to Table 6 under “Any other information on results incl. tables”.

In the urine samples, only the MOH-labelled test item groups (3 and 4) contained sufficient radioactivity for investigation. Recoveries were 88 – 102% (refer to Table 5 under “Any other information on results incl. tables”). Only two polar fractions were identified, with shorter retention times than any of the reference compounds. Parent and reference compounds were not found. For details, refer to Table 7 under “Any other information on results incl. tables”.

Table 2: Mean mass balance after a single oral administration of radiolabelled test material

Label		MOH				TBPH
Dose group (mg/kg bw)		10				10				1000
Sex		male		female		male		female		male		female
Test group		3		4		7		8		5		6
Compartment	Interval (h)	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Urine	0-8	0.432	0.136	0.327	0.116	0.002	0.000	0.001	0.000	0.015	0.008	0.001	0.000
	8-24	1.060	0.250	0.650	0.248	0.005	0.002	0.045	0.086	0.006	0.005	0.002	0.002
	24-48	0.189	0.062	0.103	0.044	0.002	0.001	0.001	0.000	0.004	0.002	0.001	0.000
	48-72	0.041	0.012	0.025	0.010	0.001	0 .000	<0.001	-	0.003	0.003	0.001	0.000
	72-96	0.021	0.004	0.013	0.004	<0.001	-	<0.001	-	0.006	0.003	0.001	-
	96-120	0.012	0.002	0.009	0.002	<0.001	-	<0.001	-	0.001	0.001	0.001	-
	120-144	0.009	0.002	0.006	0.002	<0.001	-	<0.001	-	0.003	0.003	0.001	-
	144-168	0.007	0.001	0.005	0.001	<0.001	-	<0.001	-	0.002	0.001	<0.001	-
Subtotal		1.770	0.370	1.140	0.420	0.010	0.004	0.047	0.087	0.040	0.025	0.006	0.003
Faeces	0-24	89.2	3.3	83.1	5.2	89.2	5.9	90.5	3.6	90.6	2.0	83.2	9.7
	24-48	3.16	1.67	6.89	3.30	7.10	4.39	4.71	1.74	8.89	2.10	13.50	9.20
	48-72	0.391	0.572	0.301	0.217	0.198	0.112	0.118	0.020	0.615	0.339	0.703	1.119
	72-96	0.068	0.053	0.196	0.328	0.046	0.020	0.038	0.019	0.169	0.173	0.027	0.034
	96-120	0.033	0.008	0.013	0.008	0.024	0.008	0.929	1.814	0.148	0.102	0.016	-
	120-144	0.021	0.007	0.006	0.002	0.018	0.006	0.018	0.005	0.086	0.062	0.008	-
	144-168	0.016	0.004	0.005	0.001	0.014	0.005	0.016	0.004	0.080	0.050	0.012	-
Subtotal		92.9	2.5	90.5	2.3	96.6	1.7	96.3	2.3	101.0	1.0	97.5	2.4
Cage wash		0.013	0.005	0.018	0.012	0.002	0.001	0.008	0.014	0.062	0.044	0.002	0.000
Total excreted		94.7	2.5	91.7	2.1	96.6	1.7	96.3	2.36	101.0	1.0	97.5	2.4
Liver		0.044	0.012	0.016	0.004	0.037	0.012	0.052	0.011	0.008	0.003	0.006	0.001
Kidney		0.014	0.005	0.010	0.003	0.002	0.001	0.002	0.001	<0.005	-	<0.001	-
Organs/tissues		0.059	0.016	0.027	0.007	0.056	0.017	0.075	0.017	0.007	0.005	0.006	0.001
Carcass		0.031	0.007	0.016	0.002	0.097	0.026	0.114	0.020	0.252	0.253	0.047	0.055
Intestinal tract contents		0.006	0.002	0.002	0.000	0.006	0.002	0.006	0.001	0.009	0.007	0.002	0.001
Subtotal		0.065	0.025	0.029	0.010	0.159	0.045	0.195	0.037	0.268	0.263	0.056	0.057
Total		94.8	2.5	91.7	2.1	96.7	1.7	96.5	2.4	101.0	1.0	97.5	2.4

Table 3: Distribution in tissues [µg eq/g]

Label	MOH				TBPH
Dose group (mg/kg bw)	10				10				1000
Sex	male		female		male		female		male		female
Test group	3		4		7		8		5		6
Tissue	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Adrenal Glands	<0.036	-	<0.036	-	0.084	0.027	0.238	0.073	<3.461	-	7.370	5.290
Blood	0.008	-	<0.007	-	<0.007	-	0.008	-	<0.692	-	<0.692	-
Brain	<0.007	-	<0.007	-	<0.007	-	<0.007	-	<0.692	-	<0.692	-
Epididymes	<0.007	-	-	-	0.013	0.004	-	-	<0.692	-	-	-
Eyes	<0.007	-	-	-	<0.007	-	<0.007	-	<0.692	-	<0.692	-
Fat	0.011	-	0.009	-	0.031	0.006	0.033	0.009	0.867	0.059	1.580	1.140
Femur	<0.008	-	<0.008	-	0.008	-	0.009	0.002	<0.733	-	<0.733	-
Heart	<0.008	-	<0.008	-	0.013	0.003	0.019	0.007	<0.733	-	1.100	-
Kidney	0.165	0.045	0.130	0.042	0.018	0.006	0.026	0.007	<0.733	-	1.590	-
Large intestinal tract	<0.008	-	<0.008	-	0.013	0.002	0.014	0.003	<0.733	-	<0.733	-
Liver	0.089	0.021	0.035	0.008	0.072	0.025	0.117	0.032	1.380	0.689	2.13	2.04
Lung	<0.008	-	<0.008	-	0.020	0.009	0.031	0.009	0.769	-	2.000	-
Muscle	<0.007	-	<0.007	-	0.008	0.001	0.011	0.002	<0.692	-	<0.692	-
Ovaries	-	-	0.007	-	-	_	0.126	0.054	_	-	2.980	3.440
Pancreas	<0.007	-	<0.007	-	0.026	0.008	0.032	0.009	0.930	-	2.060	-
Pituitary gland	<0.072	-	<0.072	-	<0.070	-	<0.070	-	<6.921	-	<6.921	-
Plasma	<0.007	-	<0.007	-	<0.007	-	0.008	-	<0.692	-	<0.692	-
Prostat gland	<0.007	-	-	-	0.016	0.004	-	-	<0.692	-	-	-
Sciatic nerve	<0.144	-	<0.144	-	<0.141	-	<0.141	-	<13.843	-	<13.843	-
Skin backregion	<0.007	-	<0.007	-	0.011	0.001	0.016	0.002	<0.692	-	1.030	-
Small intestinal tract	0.011	0.002	0.008	0.000	0.023	0.006	0.024	0.008	<0.692	-	1.200	-
Spinal cord	<0.007	-	<0.007	-	<0.007	-	<0.007	-	<0.692	-	<0.692	-
Spleen	<0.008	-	<0.008	-	0.024	0.010	0.030	0.008	<0.779	-	2.120	-
Stomach	<0.007	-	<0.007	-	0.014	0.005	0.018	0.004	<0.692	-	1.300	-
Testes	<0.007	-	-	-	0.010	0.002	-	-	<0.692	-	-	-
Thymus	<0.007	-	<0.007	-	0.015	0.005	0.017	0.004	<0.692	-	0.928	-
Thyroid gland	0.030	0.008	0.030	0.004	0.038	0.013	0.045	0.017	3.640	0.880	3.430	1.160
Urinary bladder	<0.072	-	<0.072	-	<0.070	-	<0.070	-	<6.921	-	<6.921	-
Uterus	-	-	<0.072	-	-	-	<0.070	-	-	-	<6.921	-
< below lower limit of quantification

Table 4: Recovery of radioactivity from the faeces

Label	MOH				TBPH
Dose group (mg/kg bw)	10				10				1000
Sex	male		female		male		female		male		female
Test group	3		4		7		8		5		6
Tissue	% in faeces	% of dose	% in faeces	% of dose	% in faeces	% of dose	% in faeces	% of dose	% in faeces	% of dose	% in faeces	% of dose
0-24 h	92	82	87	73	85	75	88	73	93	83	88	73
24-48 h	97	3	115	8	94	5	103	7	94	5	103	7

Table 5: Recovery of radioactivity from the urine of rats administered 10 mg/kg bw test item labelled with MOH

Label	MOH
Dose group (mg/kg bw)	10
Sex	male		female
Test group	3		4
Tissue	% in urine	% of dose	% in urine	% of dose
0-8 h	88	0.38	100	0.32
8-24 h	92	0.98	102	0.67

Table 6: Metabolites identified in the faeces

Label			MOH		TBPH
Dose group (mg/kg bw)			10		10		1000
Sex			male	female	male	female	male	female
Test group			3	4	7	8	5	6
Fraction	Designation	Retention time	0-48 h	0-48 h	0-48 h	0-48 h	0-48 h	0-48 h
F1	Parent compound	23.2-25.0	3.6	6.3	17.0	23.3	65.3	61.6
F2		20.9-23.3	3.6	3.9	3.1	3.1	<0.05	0.4
F3		20.1-22.4	1.2	1.0	-	-	0.5	0.5
F4	NP-2018	18.8-21.1	70.6	63.9	51.8	45.3	14.1	11.8
F5		17.5-18.9	1.9	1.6	1.1	1.0	0.6	0.4
F6	NP-1907/TBPH	16.6-18.1	<0.05	-	2.0	1.2	1.2	1.0
F7	NP-2017	11.8-13.0	-	-	8.5	4.9	3.1	3.8
F8		11.8	-	-	0.4	-	-	-
F9		10.0-11.1	-	-	1.0	0.6	-	-
F10		8.1-9.1	0.1	0.1	-	-	-	-
F11	MOH	6.3-7.1	3.6	2.4	-	-	-	-
F12		4.9-5.5	0.1	<0.05	-	-	-	-
F13		4.6	<0.05	<0.05	-	-	-	-
F14		2.9	0.2	0.1	-	-	-	-
F15		2.4	<0.05	0.1	-	-	-	-
#: sum of 0 - 24 h and 24 - 48 h sampling

Table 7: Metabolites identified in the urine of rats administered 10 mg/kg bw test item labelled with MOH

Sex		male				female
Test group		3				4
Fraction	Retention time	% in sample		% of dose		% in sample		% of dose
		0-8 h	8-24 h	0-8 h	8-24 h	0-8 h	8-24 h	0-8 h	8-24 h
U2	2.5-2.6	58.6	68.0	0.2	0.7	83.9	62.9	0.3	0.4
U1	3.2-3.3	41.4	32.0	0.2	0.3	16.2	37.1	0.1	0.2

Description of key information

The available toxicological data, the physico-chemical properties and the molecular weight of the substance Sumilizer GP (CAS No. 203255-81-6) suggest limited systemic absorption via the oral, dermal and inhalation route. Experimental data on absorption, distribution, metabolism and excretion (ADME) in rats is available for the oral route only (RCC Ltd., 2008). In this study on mass balance, metabolism and distribution, the administered radioactivity was completely recovered in excreta, organs, tissues, carcass and in the cage wash. The mass balance in all groups ranged between 91.7-101%. As the main excretion route was via the faeces (>90% of the dose for all test groups), the extent of oral absorption could not be determined.

Based on the physico-chemical properties, the substance is expected to be absorbed by micellar solubilisation. In the GI tract the substance may undergo hydrolysis and may be metabolised in the liver, as well as by microbial metabolism in the intestine. Due to the lipophilic properties of the test substance, the substance was expected to have a bioacummulation potential. This was, however, not confirmed by the ADME study in rats (Sumitomo Chemical, 2008). In this study, male and female rats were administered test item radiolabelled either at the MOH or the TBPH moiety. The major part of the administered dose (>90%) was excreted via the faeces. This was observed for both dose groups (10 and 1000 mg/kg bw/day). 0.065 - 0.27% of the dose were found in the organs, tissues, carcass and the content of the gastrointestinal tract. The highest amounts were found in the adrenal glands, liver, thyroids, ovaries and in fat. The major excretion route was via the faeces (90.5 - 101% of the dose). There was no difference between males and females. Urinary excretion accounted for ≤ 1.77% of the dose. More than 90% of the dose was excreted within 48 h post dosing.

Based on the available toxicological data and physicochemical properties, metabolites are expected to be distributed in the body and excreted mainly via the bile and urine. Fractions of the test substance which are not metabolised in the GI tract are expected to be excreted via the faeces.

In the ADME study in rats after oral exposure, a total of 15 metabolite fractions was identified in the faeces. The main fraction was attributed to reference NP-2018 (equivalent to the parent substance oxidised at the P atom by phase I metabolism).

Based on physico-chemical properties of the test item, in case of dermal uptake the substance is expected to penetrate the stratum corneum but not penetrate the viable epidermis and may be sloughed off with skin cells.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - dermal (%):

10

Additional information

Physico-chemical properties

Sumilizer GP (CAS No. 203255-81-6) is a mono-constituent substance based on analytical characterisation. The substance is a solid white powder with a molecular weight of 600.92 g/mol. The partition coefficient log Pow has been estimated to be 16.5 (QSAR calculation, KOWWIN© Version 1.6, Tognucci, 2002); the calculated vapour pressure is 6.4E-18 Pa at 20 °C (OECD 104, Calculation: Modified Watson, Tognucci, 2002 Correction). The substance was not stable in water, therefore no information on water solubility are available.

 

Basic toxicokinetics

Two types of information in toxicokinetic behaviour of Sumilizer GP are available. On the one hand, the absorption, distribution, metabolism and excretion of Sumilizer GP via the oral route were investigated in a mass balance study in rats (Sumitomo Chemical, 2008). Purpose was the comparison of the excretion balance after administration of 2 different labels, one using the radiolabel at the MOH moiety of the substance and the other one using the radiolabel in the TBPH moiety of the substance.

To address the toxicokinetic behaviour of the test item via the dermal and inhalation route, an assessment of the toxicokinetic behaviour of Sumilizer GP was conducted to the extent that can be derived from relevant available information on physico-chemical and toxicological properties generated for the other toxicological endpoints.

In the ADME study via the oral route (Sumitomo Chemical, 2008), groups of 4 male and 4 female rats were administered 10 mg/kg bw of the MOH-labelled test item (groups 3 (male) and 4 (female)), 10 mg/kg bw of the TBPH-labelled test item (groups 7 (males) and 8 (females)) or 1000 mg/kg bw of the TBPH-labelled test item (groups 5 (males) and 6 (females)). The test item was formulated in aqueous methyl cellulose and administered as single dose by oral gavage.

Urine and faeces were sampled at regular intervals. Blood, tissues/organs, the content of the gastrointestinal tract and carcass were sampled at sacrifice, 168 h after dosing. Radioactivity was determined by liquid scintillation counting (LSC) and metabolite fractions from the excreta were identified by high performance liquid chromatography (HPLC) using UV and radiodetection.

 

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

 

Oral

In the ADME study in male and female rats via the oral route (Sumitomo Chemical, 2008), test item radiolabelled in both positions was predominantly excreted via the faeces (90.5 – 101% of the dose). Therefore, the extent of oral absorption could not be determined. Oral absorption was therefore further addressed by the physico-chemical properties of the test item and by the remaining toxicological data available.

The smaller the molecule, the more easily it will be taken up. In general, molecular weights below 500 and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (> 1 mg/L). Lipophilic compounds may be taken up by micellar solubilisation by bile salts, but this mechanism may be of particular importance for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) which would otherwise be poorly absorbed (ECHA, 2012).

The substance is not stable in water, therefore no information on the solubility is available. The high log Pow (16.5) of the registered substance indicates that micellar solubilisation is the most likely mechanism for oral absorption. However, the high molecular weight of the test substance may complicate its absorption.

In a sub-chronic oral toxicity study in male and female Crj: CD rats, Sumilizer GP increased the absolute and relative liver weight in males at 1000 mg/kg bw/day and the relative liver weight in females at ≥ 300 mg/kg bw/day (Panapharm Laboratories, 1999). In addition, total cholesterol levels were increased in females at 1000 mg/kg bw/day. These effects indicate that oral absorption had occurred to a certain extent after long-term exposure. Following a single oral dose of 2000 mg/kg bw Sumilizer GP in the same rat strain, no systemic effects were observed (Sumitomo Chemical, 1998a).

The potential of a substance to be absorbed in the GI tract can be affected during the passage through the GI tract. Substances can undergo chemical changes in the GI fluids as a result of metabolism by GI flora, by enzymes released into the GI tract or by hydrolysis. These changes will alter the physico-chemical characteristics of the substance, which means that predictions made using the physico-chemical characteristics of the parent substance may no longer be accurate (ECHA, 2017). In the ADME study in rats (Sumitomo Chemicals, 2008), fractions of 15 metabolites were identified in the faeces of the animals. For details, please refer to the section “Metabolism” below.

In conclusion, the available experimental data, the physicochemical properties and molecular weight of Sumilizer GP suggest low to moderate oral absorption.

 

Dermal

The dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Dermal uptake is anticipated to be low, if the water solubility is < 1 mg/L; low to moderate if it is between 1-100 mg/L; and moderate to high if it is between 100-10000 mg/L. Dermal uptake of substances with a water solubility > 10000 mg/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values in the range of 1 to 4 (values between 2 and 3 are optimal) are favourable for dermal absorption, in particular if water solubility is high. For substances with a log Pow above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Log Pow values above 6 reduce the uptake into the stratum corneum and decrease the rate of transfer from the stratum corneum to the epidermis, thus limiting dermal absorption. Highly lipophilic substances (log P between 4 and 6) that come into contact with the skin can readily penetrate the lipid rich stratum corneum but are not well absorbed systemically. Although they may persist in the stratum corneum, they will eventually be cleared as the stratum corneum is sloughed off (ECHA, 2017).

The physicochemical properties (log Pow of 16.5) of the substance and the molecular weight (600.92 g/mol) are in a range suggestive of low absorption through the skin. By default, if the molecular mass is above 500 g/mol and the log Pow is outside the range [-1, 4], a value of 10% skin absorption is defined (ECHA, 2017; de Heer et at., 1999).

The available experimental data on acute dermal toxicity with Sumilizer GP in Wistar rats confirm the low systemic availability of the test substance. There was no evidence for any systemic effects and the LD50was estimated to be > 2000 mg/kg bw/day (RCC, 2003).

If a substance shows skin irritating or corrosive properties, damage to the skin surface may enhance penetration. If the substance has been identified as a skin sensitizer then some uptake must have occurred although it may only have been a small fraction of the applied dose (ECHA, 2017).

The available data on skin irritation and skin sensitisation did not show any irritating or sensitising effects in rabbits, therefore no enhanced penetration of the substance due to skin damage is expected (Sumitomo Chemical, 1998b and 1998c).

Overall, taking all available information into account, the dermal absorption potential is considered to be low.

 

Inhalation

Sumilizer GP is a solid with a low vapour pressure (6.4E-18 Pa at 20 °C). In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 µm may reach the thoracic region and those below 15 µm the alveolar region of the respiratory tract (ECHA, 2017).

The registered substance has a mass median diameter (MMAD) of 78.0 µm (Tognucci, 2003). Upon inhalation, the substance may therefore reach the thoracic region. Substances may be absorbed directly from the respiratory tract or, through the action of clearance mechanisms, may be transported out of the respiratory tract and swallowed. This means that absorption from the GI tract will contribute to the total systemic burden of substances that are inhaled (ECHA, 2017).

As no systemic toxicity was evident after acute oral exposure of the test substance (Sumitomo Chemical, 1998a), the absorption of the test substance by the oral route is expected to be low. Therefore, a low absorption is also considered likely if the test substance is inhaled.

As for oral absorption, the molecular weight and the log Pow are suggestive of micellar solubilisation.

Overall, systemic availability is considered unlikely after inhalation of the test substance.

 

Distribution and accumulation

Distribution of a compound within the body depends on the physicochemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration, particularly in fatty tissues (ECHA, 2017).

In the ADME study in rats (Sumitomo Chemicals, 2008) at the time of sacrifice, residues in the tissues were generally low. In the animals administered 10 mg/kg bw of the MOH-labelled test item, the highest concentrations were found in the kidneys (0.130 – 0.165 µg eq/g) and in the liver (0.035 – 0.089 µg eq/g). Residues of the TBPH-labelled test item were predominantly found in the adrenal glands (0.084 – 0.238 µg eq/g, low dose only), the thyroid gland (3.43 – 3.64 µg eq/g after the high dose and 0.038 – 0-045 µg eq/g after the low dose), in the liver (1.38 – 2.13 and 0.072 – 0.117 µg eq/g after the high and low dose, respectively), in the fat (0.87 – 1.58 µg eq/g after the high dose and 0.031 – 0.033 µg eq/g after the low dose) and in the ovaries (2.98 and 0.126 µg eq/g after the high and the low dose, respectively). There were no marked differences between the low and the high dose group.

Based on the physicochemical properties of the test substance (log Pow of 16.5) and it’s molecular weight (600.92 g/mol), the substance is taken up by micellar solubilisation and likely to distribute into cells. For substances with a log Pow > 0, the intracellular concentration may be higher than extracellular concentration, particularly in fat tissue (ECHA, 2017). Highly lipophilic substances tend in general to concentrate in adipose tissue, and depending on the conditions of exposure may accumulate within the body. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, it is generally accepted that substances with high log Pow values have long biological half-lives. The high log Pow value of 16.5 indicates that Sumilizer GP may have the potential to accumulate in adipose tissue (ECHA, 2017). This was, however, not confirmed in the oral mass balance study in rats. In males and females, more than 90% of the radioactivity was excreted within the first 48 h post dosing. For details, please refer to the section “Excretion” below.

 

Metabolism

In the oral ADME study in rats (Sumitomo Chemicals, 2008), a total of 15 metabolite fractions was detected in the faeces. Five fractions could be attributed to unchanged parent and reference substances, 10 metabolites remained unknown. The major metabolite F4 was attributed to reference compound NP-2018 (equivalent to the parent substance oxidised at the P atom by phase I metabolism), which accounted for 45 - 71% of the low dose (MOH and TBPH label) and 12 - 14% of the high dose (TBPH label). Unchanged parent compound was identified in the faeces of both low dose groups (3.6 – 6.3% for the MOH label and 17 – 23% TBPH label) and of the high dose group (62-65%, THBP label). Urine fractions were only examined for the MOH-label but not sufficient for the TBPH label groups. Two polar fractions were identified in males and females of the 10 mg/kg bw MOH-label groups, but could not be assigned to the parent or any of the reference substances.

In addition to the experimental results obtained in the ADME study in rats, potential metabolites following enzymatic metabolism of the test substance were predicted using the QSAR OECD toolbox v3.3 (OECD, 2014). This QSAR tool predicts which metabolites of the test substance may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the GI tract. Simplified, six hepatic metabolites and 5 skin metabolites were predicted for Sumilizer GP. In addition, up to 43 metabolites were predicted to result from microbiological metabolism. The predicted metabolites contain for example hydroxyl groups or carboxyl groups at various positions, which increase the test items water solubility for excretion.

The results of a guinea pig maximisation test give no indication that Sumilizer GP is activated to a reactive molecule (Sumitomo Chemical, 1998d). In addition, the test item is not activated to mutagenic intermediates under the relevant test conditions. All three experimental studies performed on genotoxicity (Ames test, chromosome aberration test and mouse lymphoma assay) were negative, with and without metabolic activation (Sumitomo Chemical, 1998e, RCC, 2005 and Sumitomo Chemical, 1999).

 

Excretion

Sumilizer GP is a highly lipophilic substance. Upon ingestion, the main route for excretion of hydrophobic substances with a high molecular weight is via the bile and subsequently the faeces. In addition, highly lipophilic substances that have penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with skin cells (ECHA, 2017).

Possible metabolites from phase I and/or phase II metabolism in the liver or from microbial metabolic processes in the intestine may be conjugated with e.g. glutathione to form more water-soluble compounds which can be excreted via the urine. The fraction of the test substance that is not absorbed in the GI tract and not accumulating in adipose tissue, is expected to be excreted via the faeces.

 In the oral ADME study in rats (Sumitomo Chemicals, 2008), radiolabelled test item (labels in both moieties) were predominantly excreted via the faeces (90.5 – 101% of the dose). Urinary excretion accounted for 1.14 – 1.77% of the dose for the MOH label and for < 0.05% of the dose or less for the TBPH label. More than 90% of the radioactivity was excreted within the first 48 h post dosing.

 

References:

 

ECHA (2017). Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance. Version 3.0.

 

OECD (2014). (Q)SAR Toolbox v3.3. Developed by Laboratory of Mathematical Chemistry, Bulgaria for the Organisation for Economic Co-operation and Development (OECD). Prediction performed 21 June 2016.http://toolbox.oasis-lmc.org/?section=overview

 

De Heer C, Wilschut A, Stevenson H and Hakkert BC (1999) Guidance document on the estimation of dermal absorption according to a tiered approach: an update. V98.1237. 1999. Zeist, NL, TNO