Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 May - 25 Aug 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

adopted 13 Apr 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministerial Des Produits Chimiques

Radiolabelling:

yes

Remarks:

14C labelled test substance (Sulfonylbenzamide-ring)

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin:
Human Skin: dermatomed human skin from 6 different donors was obtained from a recognised Tissue Bank (supplier: Biopredic, Rennes, France) and stored at approximately - 20°C prior to use. Skin sample thickness ranged between 454 and 554 µm.

Rat Skin: male Wistar Rj: WI (IOPS HAN) supplied by R. Janvier (France).
After an acclimatisation period each animal was killed by cervical dislocation and a dorsal area of the skin was clipped and this area removed for use in the study. The skin was dermatomed by use of a mini-dermatome (Decadermatome microsystem motor, Thackray Surgery, Leeds, UK) to obtain samples of 440 – 530 µm in thickness.

- Justification of species:
Human: human skin has been chosen as the human is the species of interest and for comparative purposes with the results obtained for rat skin.

Rat: chosen because of large amount of background data available concerning dermal penetration in this species and its acceptability for submission to regulatory authorities.

- Solubility in the receptor fluid:
Solubility was investigated in the receptor fluid. A volume corresponding to the maximum quantity of test substance applied to the cell, was dissolved in approximately 3 mL of the receptor fluid (Eagle's medium supplemented with 5% bovine serum albumin and gentamycin (50 mg/L)), corresponding to the total volume of the cell. This procedure simulated the conditions of maximum absorption through the skin. Samples were left for at least 24 hours. Thereafter, the samples were centrifuged (3000 rpm) for 10 minutes. Three aliquots were analysed by Liquid Scintillation Counting (LSC).

For details on the study design, see field "Any other information on materials and methods incl. tables".

Total recovery:

See Tables 1 and 2 (Any other information on results incl. tables)

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

225 mg/mL

Parameter:

percentage

Absorption:

0.85 %

Remarks on result:

other: human skin

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.27 mg/mL

Parameter:

percentage

Absorption:

1.5 %

Remarks on result:

other: human skin

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

225 mg/mL

Parameter:

percentage

Absorption:

1.76 %

Remarks on result:

other: rat skin

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.27 mg/mL

Parameter:

percentage

Absorption:

4.11 %

Remarks on result:

other: rat skin

Conversion factor human vs. animal skin:

High dose formulation:
The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours was 0.85% and 1.76% for the human and rat skin, respectively, yielding a factor difference of 2.1 between the two species for the neat product.

Low dose formulation:
The mean percentage total potentially absorbable was 1.50% and 4.11% for the human and rat skin, respectively, yielding a factor difference of 2.7 between the two species for the spray dilution.

Radiochemical Purity
The radiochemical purity of the [14C]-radiolabeled test material was confirmed for the two formulations (code samples SYP12920 and SYP12922) before starting the study. The radiochemical purity records were similar to the sample certificate values provided by the formulation supplier (>98% for both SYP12920 and SYP12922).

Solubility of test material in the receptor fluid
The solubility of [14C]-radiolabeled test material in the receptor fluid after 24 hours of incubation was demonstrated at a concentration corresponding to the maximum quantity applied on the skin dissolved in the cell volume.

Dose homogeneity

Five aliquots of the dose formulations (high dose formulation = SYP12920 and low dose formulation = SYP12922) were taken before application in order to verify the homogeneity of the dose formulations. According to the results obtained from these aliquots, the dose formulations were considered to be homogeneous.

Acheived doses

The application rate of the two formulations (SYP12920 and SYP12922) was 10 µl/cm2 skin (area of application = 1 cm2).
Therefore according to the actual specifications of each formulation, the theoretical values in terms of radiolabelled and non-radiolabelled test material were as follows:

Theoretical quantities applied on each cell:	High dose formulation (SYP12920)	Low dose formulation (SYP12922)
Radiochemical dose	49.9 kBq	11.5 kBq
Compound dose	2.25 mg	2.7 µg

Membrane integrity and membrane selection

Trans-Epidermal Water Loss (TEWL) values are attached (Appendix B, Tables B.1 and B.2). According to the variations of the environmental conditions in the experimental room (relative humidity & temperature), some differences were detected for cells from the same donor. Therefore, TEWL was used as an indicator of skin membrane integrity in conjunction with the absorption profile.

Distribution of radioactivity

High dose formulation (SYP12920):

The human skin data used to investigate the distribution of radioactivity following single dermal application of the high dose formulation was taken from 5 cells (H01 to H04 and H06) which represented dermatomed skin taken from 5 subjects.

The rat skin data used to investigate the distribution of radioactivity following single dermal application of the high dose formulation was taken from 6 cells (R01 to R06) which represented dermatomed skin taken from 6 subjects.

Table 1 presents the distribution of radioactivity for the human and rat dermatomed skin following a single topical application of the high dose formulation of [14C]-radiolabeled test material (results expressed in mean % applied radioactivity):

Table 1: [14C]-radiolabeled test material: Recovery of Radioactivity following a Single Topical Application of the test material at the High Dose in SC450 Formulation (SYP12920) to Human and Rat Dermatomed Skin Preparations.

	Test material high dose formulation (SYP12920)
	Distribution of radioactivity (% dose)
	Human skin (n = 5)		Rat Skin (n = 6)
	Mean	SD	Mean	SD
Surface dose (Tape-strips 1 & 2)	0.74	1.44	1.39	0.62
Skin Swabs (8h)	94.70	5.74	97.40	2.46
Skin swabs (24h)	0.10	0.20	0.04	0.04
Skin Swabs (8h + 24h)	94.79	5.54	97.45	2.43
Dose remaining in donor chamber	0.90	1.51	0.27	0.29
Total % non-absorbed	96.43	2.88	99.11	2.21
Skin*	0.20	0.40	0.34	0.32
Stratum Corneum^**	0.66	1.34	1.37	1.77
Total % at dose site	0.85	1.74	1.70	2.00
Receptor fluid (0 - 24h)	<0.005	<0.005	0.06	0.07
Receptor fluid terminal	<0.005	<0.005	<0.005	<0.005
Receptor chamber	<0.005	<0.005	<0.005	<0.005
Total % directly absorbed	<0.005	<0.005	0.06	0.07
Total % potentially absorbable^***	0.85	1.74	1.76	2.01
Total % recovery^****	97.29	1.58	100.87	1.40

*: skin after tape-stripping procedure
**: tape-strips excluding number 1 & 2 which are considered to be non-absorbed dose
***: including total % directly absorbed + total % at dose site
****: total recovery is issued from Debra 5.2 software calculations
SD: standard deviation
n: number of skin cells used for calculation

Human cells:
Absorption profiles are attached (Appendix E (Figure E.1). The details (per cell) are given in Appendix F (Table F.1).

Following the application of the high dose formulation of [14C]-radiolabeled test material to human skin, the overall mean recovery of the dose was 97.29%.

The mean percentage dose recovered in skin swabs at 8 hours and 24 hours (including surrounding swabs) were 94.70% and 0.10%, respectively. The mean amount retrieved from the donor chamber was 0.90% dose. In the first two tape-strips of the stratum corneum, corresponding to the surface dose, the mean amount of radioactivity was 0.74% of the dose. The mean total amount of radioactivity non-absorbed through human skin accounted for 96.43% of the applied dose.

The mean level of radioactivity remaining in the skin after tape-stripping of the treated area (including the surrounding skin) was 0.20%. The mean level of radioactivity remaining in the stratum corneum was 0.66%. The mean total of radioactivity at dose site accounted for 0.85% of the applied dose.

The mean total amount of radioactivity directly absorbed through human skin accounted for <0.005% of the applied dose, which corresponds to the sum of the mean dose recovered in the receptor fluid between 0 and 24 hours (<0.005%) and at termination (<0.005%) and the mean dose retrieved in the receptor chamber (<0.005%). Absorption profile of cell H06 appeared irregular. However, this irregularity was considered to be due to the very low levels of radioactivity recovered in the receptor fluid at each time point.

The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours from the high dose formulation was 0.85% for the human skin.

Rat cells:

Absorption profiles are presented in Appendix E (Figure E.2). The details (per cell) are given in Appendix F (Table F.2).

Following the application of the high dose formulation of [14C]-radiolabeled test material to rat skin, the overall mean recovery of the dose was 100.87%.

The mean percentage dose recovered in skin swabs at 8 hours and 24 hours (including surrounding swabs) were 97.40% and 0.04%, respectively. The mean amount retrieved from the donor chamber was 0.27% dose. In the first two tape-strips of the stratum corneum, corresponding to the surface dose, the mean amount of radioactivity was 1.39% of the dose. The mean total amount of radioactivity non-absorbed through rat skin accounted for 99.11% of the applied dose.

The mean level of radioactivity remaining in the skin after tape-stripping of the treated area (including the surrounding skin) was 0.34%. The mean level of radioactivity remaining in the stratum corneum was 1.37%. The mean total of radioactivity at dose site accounted for 1.70% of the applied dose.

The mean total amount of radioactivity directly absorbed through rat skin accounted for 0.06% of the applied dose, which corresponds to the sum of the mean dose recovered in the receptor fluid between 0 and 24 hours (0.06%) and at termination (<0.005%) and the mean dose retrieved in the receptor chamber (<0.005%).

The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours from the high dose formulation was 1.76% for the rat skin.

Low dose formulation (SYP12922):

The human skin data used to investigate the distribution of radioactivity following single dermal application of the low dose formulation was taken from 6 cells (H07 to H12) which represented dermatomed skin taken from 6 subjects.

The rat skin data used to investigate the distribution of radioactivity following single dermal application of the low dose formulation was taken from 5 cells (R07 to R11) which represented dermatomed skin taken from 5 subjects.

Table 2 presents the distribution of radioactivity for the human and rat dermatomed skin following a single topical application of the low dose formulation of [14C]-radiolabeled test material (results expressed in mean % applied radioactivity):

Table 2: [14C]-radiolabeled test material: Recovery of Radioactivity following a Single Topical Application of Test Material at the Low Dose in SC450 Formulation (SYP12922) to Human and Rat Dermatomed Skin Preparations

	Test Material low dose formulation (SYP12922)
	Distribution of radioactivity (% dose)
	Human skin (n = 6)		Rat Skin (n = 5)
	Mean	SD	Mean	SD
Surface dose (Tape-strips 1 & 2)	0.40	0.28	4.08	2.19
Skin Swabs (8h)	94.01	1.85	87.58	3.30
Skin swabs (24h)	0.05	0.05	0.35	0.21
Skin Swabs (8h + 24h)	94.06	1.85	87.93	3.43
Dose remaining in donor chamber	0.25	0.40	0.07	0.15
Total % non-absorbed	94.71	2.12	92.08	3.61
Skin^*	1.03	0.95	0.93	0.26
Stratum Corneum^**	0.38	0.25	2.75	1.78
Total % at dose site	1.41	1.04	3.68	1.62
Receptor fluid (0 - 24h)	0.08	0.14	0.42	0.25
Receptor fluid terminal	<0.005	<0.005	0.01	0.01
Receptor chamber	<0.005	<0.005	<0.005	<0.005
Total % directly absorbed	0.08	0.14	0.43	0.26
Total % potentially absorbable^***	1.50	1.11	4.11	1.73
Total % recovery^****	96.21	2.96	96.19	3.25

*: skin after tape-stripping procedure

**: tape-strips excluding number 1 & 2 which are considered to be non-absorbed dose

***: including total % directly absorbed + total % at dose site

****: total recovery is issued from Debra 5.2 software calculations SD: standard deviation

n: number of skin cells used for calculation

Human cells:
Absorption profiles are attached (Appendix E: Figure E.3). The details (per cell) are given in Appendix F (Table F.3).

Following the application of the low dose formulation of [14C]-radiolabeled test material to human skin, the overall mean recovery of the dose was 96.21%.

The mean percentage dose recovered in skin swabs at 8 and 24 hours was 94.01% and 0.05%, respectively whilst the mean amount retrieved from the donor chamber was 0.25%. In the first two tape-strips of the stratum corneum, corresponding to the surface dose, the mean amount of radioactivity corresponded to 0.40% dose. The mean total amount of radioactivity nonabsorbed through human skin accounted for 94.71% of the applied dose.

The mean level of radioactivity remaining in the skin after tape-stripping of the treated area (including the surrounding skin) was 1.03%. The mean level of radioactivity remaining in the stratum corneum was 0.38%. The mean total of radioactivity at dose site accounted for 1.41% of the applied dose.

The mean total amount of radioactivity directly absorbed through human skin accounted for 0.08% of the applied dose, which corresponds to the sum of the mean dose recovered in the receptor fluid between 0 and 24 hours (0.08%) and at termination (<0.005%) and the mean dose retrieved in the receptor chamber (<0.005%).

The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours from the low dose formulation was 1.50% for the human skin.

Rat cells:
Absorption profiles are attached (Appendix E: Figure E.4). The details (per cell) are given in Appendix F (Table F.4).

Following the application of the low dose formulation of [14C]-radiolabeled test material to rat skin, the overall mean recovery of the dose was 96.19%.

The mean percentage dose recovered in skin swabs at 8 and 24 hours were 87.58% and 0.35%, respectively whilst the mean amount retrieved from the donor chamber was 0.07%. In the first two tape-strips of the stratum corneum corresponding to the surface dose the mean amount of radioactivity corresponded to 4.08% dose. The mean total amount of radioactivity non-absorbed through rat skin accounted for 92.08% of the applied dose.

The mean level of radioactivity remaining in the skin after tape-stripping of the treated area (including the surrounding skin) was 0.93%. The mean level of radioactivity remaining in the stratum corneum was 2.75%. The mean total of radioactivity at dose site accounted for 3.68% of the applied dose.

The mean total amount of radioactivity directly absorbed through rat skin accounted for 0.43% of the applied dose, which corresponds to the sum of the mean dose recovered in the receptor fluid between 0 and 24 hours (0.42%) and at termination (0.01%) and the mean dose retrieved in the receptor chamber (<0.005%).

The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours from the low dose formulation was 4.11% for the rat skin.

Discussion of results

The distribution patterns of radioactivity were investigated following a single application of [14C]-radiolabeled test material in the SC450 formulation applied at two concentrations: a high dose formulation (nominal value = 225 mg test material/mL formulation) corresponding to the neat product, and a low dose formulation (nominal value = 0.27 mg test material/mL formulation) corresponding to a representative spray dilution.

Good recovery data were obtained for all samples, with mean total recoveries of radioactivity in the range of 96.19% to 100.87% of the applied dose.

For the high dose formulation in human and rat skin, the majority of the radioactivity was removed by swabbing (94.79% and 97.45% dose for human skin and rat skin, respectively), and by removal of the surface tape-strips (0.74% and 1.39% dose for human skin and rat skin, respectively).

For the low dose formulation, the majority of the radioactivity was removed by swabbing (94.06% and 87.93% dose for human skin and rat skin, respectively), and by removal of the surface tape-strips (0.40% and 4.08% dose for human skin and rat skin, respectively).

Since the swabbing procedure was intended to reflect a simple washing regimen at the end of the working day, the amount of radioactivity retrieved in this compartment was considered to be non-absorbed. Since the material recovered in the surface tape-strips is assumed to be associated with surface residues following incomplete removal of the dose after an 8-hour exposure period and/or material from the superficial stratum corneum, the amount of [14C]-radiolabeled test material retrieved in this compartment was considered to be non-absorbed.

Based on these results, the mean total amount of radioactivity considered as non-absorbed for the high dose formulation was 96.43% and 99.11% dose in the human and rat skin, respectively. For the low dose formulation, the mean total amount of radioactivity considered as non-absorbed was 94.71% and 92.08% dose in the human and rat skin, respectively.

The mean amount of radioactivity recovered in the skin (after tape-stripping of the treated area) accounted for 0.20% and 0.34% of the applied dose for the high dose formulation (human and rat skin, respectively). For the low dose formulation, the mean amount of radioactivity recovered in the skin accounted for 1.03% and 0.93% of the applied dose, respectively in the human and rat skin.

The mean quantity of radioactivity recovered in the stratum corneum accounted for 0.66% and 1.37% of the applied dose (human and rat skin, respectively) for the high dose formulation. For the low dose formulation, means of 0.38% and 2.75% of the applied dose were recovered in the stratum corneum (human and rat skin, respectively).

The overall mean amount of [14C]-radiolabeled test material directly absorbed was represented by the radioactivity present in the receptor fluid (including receptor fluid at termination and receptor chamber). This accounted for <0.005% and 0.06% of the applied dose in the human and rat skin, respectively, using the high dose formulation. For the low concentration, the mean total amount of radioactivity directly absorbed accounted for 0.08% and 0.43% for the human and rat skin, respectively.

The radioactivity found in the skin compartment (skin and stratum corneum but excluding surface) could be considered to be potentially absorbable. Therefore, the mean total amount of radioactivity considered as potentially absorbable (= mean % directly absorbed + mean % at dose site) for the high dose formulation was 0.85% and 1.76% dose in the human and rat skin, respectively. The mean total amount of radioactivity considered as potentially absorbable for the low dose formulation was 1.50% and 4.11% dose in the human and rat skin, respectively.

Conclusions:

This in vitro dermal penetration study was conducted in accordance to OECD TG 428 and in compliance with GLP.
The dermal penetration of [14C]-radiolabeled test material in a SC450 formulation through rat and human dermatomed skin was investigated at two concentrations (nominal values = 225 and 0.27 mg/mL, for the high and low dose formulations, respectively).
For both formulations, the majority of the radioactivity applied to the skin was removed by swabbing and by removal of the surface layer of the stratum corneum and was considered to be non-absorbed.
High dose formulation:
The mean percentage of [14C]-radiolabeled test material directly absorbed over a period of 24 hours was <0.005% and 0.06% for the human and rat skin, respectively.
The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours was 0.85% and 1.76% for the human and rat skin, respectively, yielding a factor difference of 2.1 between the two species for the neat product.
Low dose formulation:
The mean percentage directly absorbed was 0.08% and 0.43% for the human and rat skin, respectively.
The mean percentage total potentially absorbable was 1.50% and 4.11% for the human and rat skin, respectively, yielding a factor difference of 2.7 between the two species for the spray dilution.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2004 - 24 Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Sulfonylbenzamide-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 50-67
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution (test substance dissolved in acetonitrile) was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 28 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Actual dose received: 4.95 mg/kg bw/day

No. of animals per sex per dose / concentration:

8 males recieved radiolabelled test substance
1 male recieved non-radiolabelled test substance (control)

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The selected dose level was based on the dosage recommendations of the current EPA Health Effects Guidelines and was in the same range as the low dose tests in the corresponding rat metabolism studies.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air
- Time and frequency of sampling:
Urine: 1, 4, 8, 24, 48, 72, 96, 120, 144 and 168 h after dosing
Faeces: 24, 48, 72, 96, 120, 144 and 168 h after dosing
Expired air: 24 and 48 hours after dosing
QBWA: 24 and 48 hours after dosing (for 4 animals)

The control animal for autoradiography of the carcass was sacrificed after 4 h.

ANALYTICAL METHOD
- Measurement of Liquid Samples Using Liquid Scintillators: The measurement of the radioactivity in liquid samples was carried out by liquid scintillation counting (LSC).
- Measurement of Solid Samples Using Liquid Scintillators: Solid faeces samples were weighed and combusted in an oxygen atmosphere using the "Oxidizer 387" (Packard Instruments). The released 14C02 was trapped in an alkaline absorber (Carbosorb E, Packard Instruments) and radio-assayed by LSC using Permafluor E+ (Packard Instruments) as scintillator.
- Quantitative Whole Body Autoradiography (Radioluminography): The digital images of the autoradiograms obtained by the Fuji BAS 5000 system were used for the assessment of the distribution of radioactivity concentrations in different organs and tissues.
- Reversed phase HPLC (Agilent HP 1100) in combination with UV / radio-detector (Ramona star) / mass spectrometry (TSQ 7000) were used for quantification as well as for co-chromatographic investigations.

Preliminary studies:

no

Type:

absorption

Results:

The radiolabelled test substance was absorbed quickly from the gastrointestinal tract. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after administration.

Type:

distribution

Results:

At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 48 h after administration (90% excretion).

Details on absorption:

One hour after the administration of [Sulfonylbenzamide-ring-UL-14C]AE 0001789, the maximum concentration of radioactivity was observed in the contents of stomach and small intestine, which is an inherent result of the oral administration. High radioactivity was also observed in blood, indicating fast absorption and high bioavailability of AE 0001789.

Details on distribution in tissues:

A high concentration of radioactivity was mainly observed in the excretory organs liver and kidney, indicating commencing clearance of the compound immediately after absorption mainly by the renal route.
The highest radioactivity concentration (Cmax) in all organs and tissues was observed 1 hour after the administration. The radioactivity levels observed in blood decreased quickly to an intermediate minimum observed in the animal sacrificed 24 hours after the administration (0.189 µg/g), before they increased again to a second maximum 48 hours after the administration (0.234 µg/g). This effect can be attributed to an interruption of the fast absorption in the intestine resulting from the delayed gastric emptying which was observed between 4 and 48 hours after administration.
Overall, the equivalent concentrations in most organs and tissues were relatively low compared to the actual administered amount of 4.95 mg/kg bw. Only the dose normalised concentrations (CN) at Cmax for renal medulla was close to the equilibrium value of 1, which is an indication for the efficient depletion of the compound via kidney and urine. The CN-values for blood, liver and renal cortex were between 0.5 and 0.7. All other CN-values were well below 0.5.

See Attachment 1 (background material) for tabulated distribution data.

Details on excretion:

The excretion of total radioactivity was almost complete 48 h after administration of 14C-AE 0001789. At this time more than 90% of the administered dose had been excreted via urine and faeces. The major part of the radioactivity administered was excreted by urine. The expiration of 14CO2 and other 14C-labelled volatiles could not be determined quantitatively due to a malfunction of the collection equipment. However, the volatile radioactivity was determined in the course of an autoradiography test with AE 0001789 labelled in the methoxybenzoyl moiety. This test demonstrated the high stability of the labelling position with regard to possible formation of volatile products (key, 2006, M-274337-01-1, rat, oral, metabolism, RL1). As no molecule cleavage was observed in the metabolism studies performed in rat (key, 2006, M-274975-01-2, rat, oral, metabolism, RL1; key, 2006, M-276053-01-2, rat, oral, metabolism, RL1) with both radiolabels, a repetition of the test for volatile radioactivity with the [sulfonylbenzamide-ring-UL-C]-label was not considered necessary.

See Attachment 2 (background material) for tabulated excretion data.

Metabolites identified:

not measured

Details on metabolites:

The metabolic profiles in urine and faeces extracts were determined by reversed phase HPLC with radio-detection. The recorded metabolic profiles are shown in Attachment 3. No isolation or structure elucidation of compounds detected in these profiles was performed in the study.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. The 14C labelled test substance (Sulfonylbenzamide-ring) was absorbed quickly from the gastrointestinal tract. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after the administration. The absorption process was apparently interrupted, probably due to delayed gastric emptying occurring between 4 and 48 hours after dosing. The high radioactivity observed in kidney already 1 hour after dosing indicated that renal excretion commenced immediately after absorption.
The absorbed radioactivity was not uniformly distributed in the body. At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.
Residues in all organs and tissues decreased rapidly between 1 and 72 hours. In all organs and tissues residues were < LOD or < LOQ at later time-points between 120 and 168 hours after dosing.There was no sign for retention of radioactivity in specific organs or tissues. Residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, or thyroid gland) were rapidly depleted in parallel with depletion from the other organs and tissues.
The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete at the end of the study period.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2004 - 24 Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Methoxybenzoyl-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: Approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25
- Humidity (%): 50-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 28 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Actual dose received: 4.94 mg/kg bw

No. of animals per sex per dose / concentration:

8 males

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The selected dose level was based on the dosage recommendations of the current EPA Health Effects Guidelines and was in the same range as the low dose tests in the corresponding rat metabolism studies.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air
- Time and frequency of sampling: 1, 4, 8, 24, 48, 72, 96, 120, 144 and 168 h after dosing (The control animal was sacrificed after 4 h).

Preliminary studies:

no

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after administration.

Type:

distribution

Results:

At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 48 h after administration (90% excretion). Examination of volatiles demonstrated high stability of the labelling position.

Details on absorption:

One hour after the administration of [methoxybenzoyl-ring-UL-14C]AE 0001789, the maximum concentration of radioactivity was observed in the contents of stomach and small intestine, which is an inherent result of the oral administration. High radioactivity was also observed in blood, indicating fast absorption and high bioavailability of AE 0001789.

Details on distribution in tissues:

A high concentration of radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.
The highest radioactivity concentration (Cmax) in all organs and tissues was observed 1 hour after the administration. The radioactivity levels observed in blood decreased quickly to an intermediate minimum observed in the animal sacrificed 8 hours after the administration (0.230 µg/g), before they increased again to a second maximum 24 hours after the administration (0.375 µg/g). This effect can be attributed to an interruption of the fast absorption in the intestine resulting from the delayed gastric emptying which was observed between 4 and 24 hours after administration.
Overall, the equivalent concentrations in most organs and tissues were relatively low compared to the actual administered amount of 4.94 mg/kg bw. Only the dose normalised concentrations (CN) at Cmax for renal medulla was significantly larger than 1, which is an indication for the efficient depletion of the compound via kidney and urine. The CN-value for blood was also slightly above 1, and the CN-values of liver and renal cortex were slightly below the equilibrium value of 1. All other CN-values were well below 1.

See Attachment 1 (background material) for tabulated distribution data.

Details on excretion:

The excretion of total radioactivity was almost complete 48 h after administration of 14C-AE 0001789. At this time more than 90% of the administered dose had been excreted via urine and faeces. The major part of the radioactivity administered was excreted by urine. The expiration of 14CO2 and other 14C-labelled volatiles was investigated with the animals sacrificed later than 24 hours. During the monitored period of 48 hours, at maximum 0.01% of the administered dose were expired as carbon dioxide or other volatiles in all cases. This demonstrates the high stability of the labelling position with regard to possible formation of volatile products.

See Attachment 2 (background material) for tabulated excretion data.

Metabolites identified:

not measured

Details on metabolites:

The metabolic profiles in urine and faeces extracts were determined by reversed phase HPLC with radio-detection. The recorded metabolic profiles are shown in Attachment 3. No isolation or structure elucidation of compounds detected in these profiles was performed in the study.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. The 14C labelled test substance (Methoxybenzoyl-ring) was absorbed quickly from the gastrointestinal tract. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after the administration. The absorption process was apparently interrupted, probably due to delayed gastric emptying occurring between 4 and 24 hours after dosing. The high radioactivity observed in kidney already 1 hour after dosing indicated that renal excretion commenced immediately after absorption.
The absorbed radioactivity was not uniformly distributed in the body. At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.
Residues in all organs and tissues decreased rapidly between 1 and 48 hours. In all organs and tissues residues were < LOD or < LOQ at later time-points between 72 and 168 hours after dosing. There was no sign for retention of radioactivity in specific organs or tissues. Residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, or thyroid gland) were rapidly depleted in parallel with the depletion from the other organs and tissues.
The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete at the end of the study period. No significant expiration of 14C-labelled volatiles was observed.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jan 2005 - 12 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Methoxybenzoyl-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: Approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; 16 g/day
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 27-40
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 16 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

2 mg/kg bw/day (actual dose received)

Remarks:

2.05 mg/kg bw (actual dose received)

No. of animals per sex per dose / concentration:

4 males

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The dose level of 2 mg/kg bw was selected based on the toxicological properties of the test substance. This dose level is in the range of the NOEL, but high enough to allow for metabolite identification in excreta.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, plasma
- Time and frequency of sampling: 4, 8, 12, 24, 48, 72 and 96 h (urine); 24, 48, 72 and 96 h (faeces); 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48, 72 and 96 h (plasma)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, tissues
- Time and frequency of sampling: 4, 8, 12, 24, 48, 72 and 96 h (urine); 24 h (faeces); at sacrifice (tissues)
- From how many animals: Samples pooled
- Method types for identification: HPLC-UV, TLC-UV, LC-MS

ANALYTICAL METHOD
HPLC/HPTLC-UV combined with a radioactivity detector and LC-MS were used for measuring radioactivity, quantitation of parent compound and/or metabolites in composite urine samples and faeces extracts, and quantitation of the test substance in the administered dose formulations.

Measurement of radioactivity
- Liquid samples: Liquid scintillation counting (LSC). Small organs or tissues (adrenal glands, thyroid, ovaries, perirenal fat,and uterus ) were solubilised by means of a tissue solubiliser (e.g.BTS-450).
- Solid samples: All samples with the exception of those dissolved in BTS were weighed and combusted in an oxygen atmosphere. The released 14C02 was trapped in an alkaline absorber and radio-assayed by LSC using a scintillator.
- The limit of detection (LOD) was established at 20 dpm measured per aliquot after correction for the background radioactivity. The limit of quantification (LOQ) for each individual measurement was established as 2 to 3 times of the background radioactivity (dpm) of each instrument/method.

Statistics:

The values were checked for outliers in accordance with the outlier test by Nalimov, if appropriate. Values identified as outliers were marked and not used for the calculations of arithmetic means and coefficients of variations (in %). In addition, the concentrations or amounts of radioactivity were checked for values below the limit of detection (LOD). Values below the LOD were not considered in calculations of arithmetic means.

Preliminary studies:

no

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in almost all organs and tissues was found approx. 40 min after administration.

Type:

distribution

Results:

No significant residues remained in the bodies of the rats at sacrifice, 96 h after administration (approx. 0.07% of the administered dose). No radioactive residues were accumulated in any of the tissues, organs or glands.

Type:

metabolism

Results:

AE 0001789 was metabolised to a low extent: Parent compound (76%), AE 0001789-descyclopropylamino (5%), AE 0001789-desmethyl (0.6%), AE 0001789-anisic acid (0.3%).

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 24 h after administration (98% excretion of the recovered dose).

Details on absorption:

The test substance was very rapidly absorbed and absorption commenced immediately after oral dosing. The maximum plasma concentration was reached approximately 40 min after the administration (tmax). Absorption was nearly complete; approximately 82% of the dose recovered was renally excreted or remained in the body at sacrifice (without gastrointestinal tract).

Details on distribution in tissues:

The maximum plasma concentration (Cmax = 4.36 µg/g) was reached approx. 40 minutes after dosage (tmax = 0.7 h) reflecting rapid absorption. Afterwards, the plasma concentrations declined to less than 1% of the maximum concentration within 72 hours post administration, indicating continuous distribution and elimination and that no retention of the compound related residues in the body of the animals took place.

At sacrifice, only approx. 0.07% of the administered dose was found in the bodies of the rats. The major fraction was detected in organs and tissues (0.04%) and a minor proportion in the gastrointestinal tract (0.01%). Among the organs and tissues, the highest residues were found in the carcass (0.03%), followed by the residues in the skin (0.02%) and liver (0.01%). All other analysed organs and tissues contained far less than 0.01% of the administered radioactivity.

Only approx. 85% of the radioactivity administered was recovered. This was probably due to losses of small amounts of urine for two animals, which contained a high radioactivity concentration.

See Attachment 4 for tabulated on radioactive residues in organs and tissues.
See Attachment 5 for tabulated data of the time course of 14C-concentration in the plasma.
See Attachment 6 for tabulated data of the pharmacokinetic parameters.

Details on excretion:

Elimination of the test substance was almost complete 24 h after administration. At this time more than 83% of the administered dose (corresponding to >98% of the recovered dose) had been excreted with urine and faeces. At the time of sacrifice, 96 h after administration, more than 85% of the administered and more than 99% of the recovered dose had been excreted. Approx. 82% of the recovered radioactivity was excreted by urine and approx. 18% was excreted by the faeces.

See Attachment 2 for tabulated data of excretion profiles - Urine and Faeces.
See Attachment 3 for a balance of radioactivity in excreta and tissues, and percentage total radioactivity recovered.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 4.36 µg/g

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 40 min (0.7 h)

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 13.43 mg/L*h

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 22.9 h

Key result

Test no.:

#1

Toxicokinetic parameters:

other: Total clearance: 2.48 mL/min

Key result

Test no.:

#1

Toxicokinetic parameters:

other: Mean residence time: 4.85 h

Metabolites identified:

yes

Details on metabolites:

AE 0001789 was metabolised to a low extent. Parent compound was the major component and represented in total approx. 76% of the administered dose. Three metabolites were detected in urine: AE 0001789-descycloprpylamino, representing approx. 5% of the administered dose and the minor metabolites AE 0001789-desmethyl and AE 0001789-anisic acid. Minor amounts of AE 0001789-descyclopropylamino were also identified in faeces, as well as minor amounts of AE 0001789-desmethyl.

The main metabolic reactions of [methoxy-benzoyl-ring-UL-14C]AE 0001789 observed in the rat are:
- Elimination of the cyclopropylamine moiety by hydrolysis of the carboxamide bond in the sulfonylbenzamide moiety to give AE 0001789-descyclopropylamino,
- Desmethylation of the methoxybenzoyl moiety results in AE 0001789-desmethyl,
- Hydrolytic cleavage of the carboxamide bond in the methoxybenzoyl moiety to form AE 0001789-anisic acid.

See Attachment 1 for the proposed metabolic pathway in rat.
See Attachment 7 for the balance of test substance and metabolites excreted with urine and faeces.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat.
A group of male rats was orally administered by gavage with a single dose of 14C labelled test substance (Methoxybenzoyl-ring) in a 0.5% aqueous Tragacanth suspension at a nominal dose level of 2 mg/kg bw. The animals were sacrificed 96 hours after administration.
The test substance was absorbed very quickly; the maximum plasma concentration was reached approx. 40 min after administration. Absorption was nearly complete in the test; approximately 82% of the dose recovered was renally excreted or remained in the body at sacrifice (without GIT).
The compound related radioactivity was quickly and completely excreted, approximately 85% of the administered dose (>99% of the recovered dose) had been excreted via urine and faeces at the time of sacrifice, 96 hours after administration. The predominant route of excretion was via urine, accounting for 70% of the dose administered, 15% was excreted with the faeces.
The test substance was efficiently eliminated from the body as illustrated by the terminal half-life of 23 h, and by the total clearance of 2.5 mL/min. More than 83% of the administered dose (98% of the recovered dose) was excreted within 24 h after administration.
Total radioactive residues in the organs and tissues were very low (<0.0073 µg/g), in all cases near or below the limit of detection.
The test substance was metabolised to a low extent. The main compound detected in urine and faeces was parent compound. AE 0001789-descyclopropylamino was formed as major metabolite by hydrolytic cleavage of the carboxamide bond in the sulfonylbenzamide moiety and the loss of the descyclopropylamine moiety. Hydrolysis of the carboxamide bond in the methoxybenzoyl moiety led to the minor label-specific metabolite AE 0001789-anisic acid. Minor metabolite AE 0001789-desmethyl resulted from desmethylation of the parent compound.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Sep 2004 - 29 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 4 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

Remarks:

14C labelled test substance (Sulfonylbenzamide-ring)

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann Versuchstierzucht GmbH, Borchen, Germany
- Age at study initiation: 7 weeks (males); 10 weeks (females)
- Weight at study initiation: approx. 200 g
- Housing: Individually in Makrolon metabolism cages.
- Individual metabolism cages: Yes
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15), supplied by Provimi Kliba AG, Switzerland; ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 44-67
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the radiolabelled and non-radiolabelled test substance an adequate aliquot of the stock solution was taken and evaporated to near dryness under a gentle stream of nitrogen. The residue was suspended in 16 mL of 0.5% aqueous Tragacanth.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Actual dose received (m/f): 2.17/2.20 mg/kg bw

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Actual dose received (m/f): 190.23/193.46 mg/kg bw

No. of animals per sex per dose / concentration:

4

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: The dose levels were selected based on the toxicological properties of the test substance. A dose level of 200 mg/kg was selected for the high dose experiments, which is sufficiently below the LD50. The low dose level was fixed at 2 mg/kg, and thus in the range of the NOEL.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, plasma
- Time and frequency of sampling: 4, 8, 24, 48, 72 and 96 h (urine; high-dosed males were additionally sampled at 12 h); 24, 48, 72 and 96 h (faeces); 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48, 72 and 96 h (plasma)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, tissues
- Time and frequency of sampling: 4, 8, 12, 24, 48, 72 and 96 h (urine); 24 h (faeces), at sacrifice (tissues)
- From how many animals: Samples pooled
- Method types for identification: HPLC-UV, TLC-UV, LC-MS

ANALYTICAL METHOD
HPLC/HPTLC-UV combined with a radioactivity detector and LC-MS were used for measuring radioactivity, quantitation of parent compound and/or metabolites in composite urine samples and faeces extracts, and quantitation of the test substance in the administered dose formulations.

Measurement of radioactivity
- Liquid samples: Liquid scintillation counting (LSC). Small organs or tissues (adrenal glands, thyroid, ovaries, perirenal fat,and uterus ) were solubilised by means of a tissue solubiliser (e.g.BTS-450).
- Solid samples: All samples with the exception of those dissolved in BTS were weighed and combusted in an oxygen atmosphere. The released 14C02 was trapped in an alkaline absorber and radio-assayed by LSC using a scintillator.
- The limit of detection (LOD) was established at 20 dpm measured per aliquot after correction for the background radioactivity. The limit of quantification (LOQ) for each individual measurement was established as 2 to 3 times of the background radioactivity (dpm) of each instrument/method.

Statistics:

The values were checked for outliers in accordance with the outlier test by Nalimov, if appropriate. Values identified as outliers were marked and not used for the calculations of arithmetic means and coefficients of variations (in %). In addition, the concentrations or amounts of radioactivity were checked for values below the limit of detection (LOD). Values below the LOD were not considered in calculations of arithmetic means.

Preliminary studies:

no

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in almost all organs and tissues was found approx. 10-60 min after administration.

Type:

distribution

Results:

No significant residues remained in the bodies of the rats at sacrifice, 96 h after administration (<0.06% of the administered dose). No radioactive residues were accumulated in any of the tissues, organs or glands.

Type:

metabolism

Results:

AE 0001789 was metabolised to a low extent: Parent compound (80-95%), AE 0001789-descyclopropylamino (3-8%), AE 0001789-desmethyl (0.17-0.4%), AE 0001789-cyclopropyl-sulfamoylbenzamide (0-0.64%).

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete 72 h after administration (98% excretion of the recovered dose).

Details on absorption:

After administration of 2 mg/kg bw AE 0001789 to male and female rats, between 90 and 91% of the recovered total radioactivity were excreted with the urine. Only between 9 and 10% of the dose were detected in the faeces and the GIT of rats.
After administration of 200 mg/kg bw AE 0001789, renal excretion was slightly lower with values between 69 and 74% of the dose recovered, although still indicating a high level of absorption at this dose level. The absorption rate (renal excretion plus residues in body without GIT), based on the low dose tests, therefore amounted to more than 90% of the dose recovered.
The absorption of AE 0001789 commenced immediately after administration as shown by the concentration of radioactivity in the plasma. See Attachment 5 for tabulated data of the time course of 14C-concentration in the plasma.

Details on distribution in tissues:

After a single oral administration of 2 mg/kg bw AE 0001789 to male and to female rats the maximum of the plasma concentration of radioactivity was reached approximately 10 to 40 minutes after dosage (tmax). The equivalent concentrations Cmax were in a similar range for males and females with approximately 5.6 µg/g.
In the high dose tests with 200 mg/kg bw AE 0001789, the maximum of the plasma concentration was observed 40 to 60 minutes after dosing. The maximum dose normalised concentrations were considerably lower than for the low dose tests for both sexes. This indicates that a saturation of the absorption process has occurred at the high dose level.
The curves obtained in the high-dose tests feature a double-peaked curve for both, male and female rats. A second plasma maximum of the mean plasma concentrations is apparent 32 hours after the administration for female rats and 48 hours after dosing for male rats. These double peak phenomena in the plasma concentration-time profile are well in line with the excretion profiles, which also show that a large proportion of compound-related radioactivity is renally excreted at later time points, after 24 hours post dose.
Double peak phenomena in plasma profiles have been described in the pharmacokinetic literature as effects caused by discontinuous absorption and/or delayed gastric emptying of a portion of the dose. In the case of AE 0001789 it is assumed, that delayed gastric emptying causes the double peak phenomena since a large proportion of compound-related radioactivity (up to 47% of the administered dose) was excreted later than 24 hours after dosing.
Apart from these observations, no significant differences were apparent with regard to the levels of the dose normalised concentrations in the high dose and in the low dose experiment. The dose normalised concentrations in the distribution/elimination phase and the decrease of the plasma levels were similar in all tests.
The mean residence time (MRT) of AE 0001789-related radioactivity was short for all tests included in the study, ranging from 5 to 26 hours.
The area under the plasma curve (AUC) was similar for males and females in the low dose tests, with 17 and 13 mg/L*h. In the corresponding high dose tests it was significantly higher, with 545 and 567 mg/L*h. The AUC-ratios of high dose versus low dose were 31 for males and 44 for females, i.e. under-proportional to the increase in dose, indicating a certain saturation of the absorption process in the high dose.
The terminal half-lives (t1/2) were calculated using the last data points, only, and were in the range of 13 to 21 hours. The half-lives were in the same order of magnitude for all tests and thus apparently unaffected by the dose levels. The total clearance (CL) was in the range of 2 to 6 mL/min. These values demonstrate the efficient elimination of the AE 0001789-related radioactivity from the rats.
At sacrifice, only between 0.02% and 0.06% of the administered dose was found in the bodies of the rats. The major fraction, in total between 0.01% and 0.04%, was detected in organs and tissues and a minor proportion in the gastro-intestinal tract. Very low dose normalised concentrations were detected in all organs and tissues in all tests. The individual values, ranged from
See Attachment 4 for tabulated on radioactive residues in organs and tissues.
See Attachment 5 for tabulated data of the time course of 14C-concentration in the plasma.
See Attachment 6 for tabulated data of the pharmacokinetic parameters.

Details on excretion:

The excretion of total radioactivity was almost complete 72 h after a single oral administration of AE 0001789. At this time more than 84% of the administered dose (corresponding to >98% of the recovered dose) had been excreted via urine and faeces in all four tests. At the time of sacrifice, 96 h after administration, more than 85% of the administered and more than 99% of the recovered dose had been excreted in all cases.
Between 79 and 90% of the administered radioactivity was excreted by urine in the low dose tests, and between 69 and 72% in the high dose tests. Faecal excretion accounted for between 7 and 10% of the administered dose in the low dose tests and between 25 and 31% in the high dose tests. The higher faecal excretion in the high dose tests was probably due to an incomplete absorption of AE 0001789 from the GIT into plasma. No sex difference was apparent in the faecal excretion rate.
The profile of excretion over time shows that a large proportion of compound-related radioactivity was excreted after 24 hours post dose in the high dose tests. Up to 47% of the administered dose was excreted per day between 24 and 72 hours after dosing. This indicates most probably delayed gastric emptying.

See Attachment 2 for tabulated data of excretion profiles - Urine and Faeces.
See Attachment 3 for a balance of radioactivity in excreta and tissues, and percentage total radioactivity recovered.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 62.6 µg/g

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 40 min (0.7 h)

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 545.18 mg/L*h

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 13 h

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 81.5 µg/g

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 1 h

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 566.69 mg/L*h

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 15.1 h

Key result

Test no.:

#3

Toxicokinetic parameters:

Cmax: 5.57 µg/g

Key result

Test no.:

#3

Toxicokinetic parameters:

Tmax: 40 min (0.7 h)

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 17.49 mg/L*h

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 21.4 h

Key result

Test no.:

#4

Toxicokinetic parameters:

Cmax: 5.57 µg/g

Key result

Test no.:

#4

Toxicokinetic parameters:

Tmax: 10 min (0.17 h)

Key result

Test no.:

#4

Toxicokinetic parameters:

AUC: 12.84 mg/L*h

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 19.5 h

Metabolites identified:

yes

Details on metabolites:

AE 0001789 was metabolised to a low extent. Parent compound was the major component and represented in total approx. 80-95% of the administered dose. Three metabolites were detected in urine: AE 0001789-descyclopropylamino (3-8%) was formed as major metabolite by hydrolytic cleavage of the carboxamide bond in the sulfonylbenzamide moiety and the loss of the descyclopropylamine moiety. Hydrolysis of the carboxamide bond in the methoxybenzoyl moiety led to the minor metabolite AE 0001789-cyclopropylsulfamoylbenzamide, which can also be formed from metabolite AE 0001789-desmethyl. AE 0001789-desmethyl resulted from desmethylation of the parent compound.

See Attachment 1 for the proposed metabolic pathway.
See Attachment 7 for the balance of test substance and metabolites excreted with urine and faeces.

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to OECD 417. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat.
Groups of 4 male and female rats were orally administered by gavage with a single dose of 14C labelled test substance (Sulfonylbenzamide-ring) in a 0.5% aqueous Tragacanth suspension at nominal dose levels of 2 and 200 mg/kg bw. The animals were sacrificed 96 hours after administration.
The test substance was rapidly absorbed in all tests, the maximum plasma concentrations were reached between approximately 10 and 60 minutes after administration. Good absorption was also detected at the high dose level, though renal excretion was slightly lower than in the low dose tests. Absorption was complete in the low dose tests, more than 90% of the dose recovered were renally excreted or remained in the body at sacrifice (without GIT).
The compound was quickly and efficiently eliminated from the body in all tests as illustrated by the short terminal half-lives (t1/2) in the range of 13 to 21 h, and by the total clearance in the range of 2 to 6 mL/min. Excretion of total radioactivity was almost complete 72 h after administration. More than 84% of the administered dose (corresponding to >98% of the recovered dose) was excreted 72 h after the administration.
Delayed gastric emptying was observed in the high dose tests, leading to double peak phenomena in the plasma concentration-time curves and late excretion of significant portions of the administered dose (up to 47% per day) between 24 and 72 h after administration.
The predominant route of excretion was via urine, accounting for 69 to 90% of the radioactivity administered. In the high dose tests, a certain saturation of the absorption process could be observed. The maximum dose-normalised concentrations in plasma were lower than in the low dose tests, and faecal excretion increased by a factor of 2 - 3 compared to the low dose tests.
No significant residues remained in the bodies of the rats at sacrifice, 96 h after administration (<0.06% of the administered dose). No radioactive residues were accumulated in any of the tissues, organs or glands.
AE 0001789-descyclopropylamino was formed as major metabolite by hydrolytic cleavage of the carboxamide bond in the sulfonylbenzamide moiety and the loss of the descyclopropylamine moiety. Hydrolysis of the carboxamide bond in the methoxybenzoyl moiety led to the minor metabolite AE 0001789-cyclopropylsulfamoylbenzamide, which can also be formed from metabolite AE 0001789-desmethyl. AE 0001789-desmethyl resulted from desmethylation of the parent compound.

Description of key information

Rapid absorption of the test substance occurred after oral exposure along the gastrointestinal tract. This was evident in rodent studies on toxicokinetics as well as the repeated-dose toxicity studies due to observed systemic effects. The distribution of test substance-related radioactivity within the body was uniform. The quantity of radioactivity in all organs and tissues was low compared to administered dose and always lower than the level in blood, indicating no tendency of bioaccumulation. The highest residue levels were detected in the kidney (renal medulla), acting as main organ for excretion. The test compound underwent minimal metabolism in rodents and the elimination from the organism was fast. There is no indication of any bioaccumulation potential of the parent compound and/or its metabolites. With regard to dermal absorption, an in vitro study indicated low potential for dermal penetration for the substance. This was supported by the lack of any effects in the acute dermal study.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

1.5

Absorption rate - inhalation (%):

100

Additional information

Summary of the available toxicokinetic studies

In order to determine the distribution of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide, male rats were orally administered with a target dose of 5 mg/kg bw of radioactive test substance, either uniformly labelled with 14C in the sulfonylbenzamide- or in the methoxybenzoyl-ring of the molecule, in two independent studies (M-274287-01-1 and M-274337-01-1, respectively). After an observation period of up to 168 hours quantitative whole body autoradiography (QWBA) was performed.

In both studies the test substance was absorbed quickly from the gastrointestinal tract and featured high bioavailability. The maximum concentration of radioactivity in almost all organs and tissues was found 1 hour after the administration. The absorption process was apparently discontinuous, probably due to delayed gastric emptying occurring between 4 and 48 hours after dosing. The high radioactivity observed in kidney already 1 hour after dosing indicated that renal excretion commenced immediately after absorption.

The absorbed radioactivity was not uniformly distributed in the body. At all time-points examined, high radioactivity was mainly observed in the excretory organs kidney and liver. The residues in all other organs and tissues were fairly evenly distributed and always lower than the residues observed in blood.

Residues in all organs and tissues decreased rapidly between 1 and 72 hours. In all organs and tissues residues were below the limit of detection (LOD) or quantification (LOQ) at later time-points between 72 and 168 hours after dosing. There was no sign for retention of radioactivity in specific organs or tissues. Residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, or thyroid gland) were rapidly depleted in parallel with depletion from the other organs and tissues. The test substance was rapidly eliminated from the body, predominantly via renal excretion. Excretion was almost complete at the end of the study period. No significant expiration of 14C-labelled volatiles was observed.

In addition, the biokinetic behaviour and metabolism of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide was investigated in two separate studies in the rat using either the [sulfonylbenzamide-ring-UL-14C]- or the [methoxybenzoyl-ring-UL-14C]-labelled compound (M-276053-01-1 and M-274975-01-1, respectively). In the first study performed with the [sulfonylbenzamide-ring-UL-14C]-labelling a single high dose of 200 mg/kg bw and a single low dose of 2 mg/kg bw test substance were administered to male and female rats. Since no significant amounts of label-specific metabolites were formed and no significant sex differences were apparent, no tests with female rats or high dose levels were included in the study using the compound radiolabelled in the methoxybenzoyl moiety.

The absorption of the test substance was fast in all tests and commenced immediately after oral administration. The maximum plasma concentration was reached within 1 hour after administration. Double peak phenomena in the plasma profiles of the high dose tests and a discontinuous excretion pattern indicated delayed gastric emptying of a portion of the dose. Absorption in the low dose tests was nearly complete; 82% and more of the dose recovered was renally excreted or remained in the body at sacrifice (without gastrointestinal tract).

The distribution of the test substance into organs and tissues was followed using plasma kinetics. The plasma concentrations in all tests declined to less than 1% of the maximum concentration within 72 hours post administration, indicating that no retention of the compound related residues in the body of the animals took place.

Elimination of the test substance was rather fast and efficient in all tests as illustrated by the short terminal half-lives of the radioactive residues in the plasma in the range of 13 to 23 h. Nevertheless, large proportions of compound-related radioactivity were excreted 24 hours after administration in the high dose tests indicating delayed gastric emptying. Residues in tissues and organs at sacrifice were very low.

N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide was metabolised only to a low extent. The parent compound (AE 0001789) was the main component detected in urine and faeces, accounting in all tests for more than 76% of the dose administered. Independent of the radiolabelled position, AE 0001789-descyclopropylamino was the major metabolite detected (2.37-7.93% of administered dose). Furthermore, the minor metabolite AE 0001789-desmethyl was verified in all tests. The second minor metabolite differed, depending on whether the test substance was radiolabelled in the methoxybenzoyl or the sulfonylbenzamide moiety. Administration of [sulfonylbenzamide-ring-UL-14C]-AE 0001789 resulted in AE 0001789-cyclopropylsulfamoylbenzamide, whereas administration of [methoxybenzoyl-ring-UL-14C]-AE 0001789 lead to AE 0001789-anisic acid.

The main metabolic reactions of AE 0001789 observed in the rat are:

- Elimination of the cyclopropylamine moiety by hydrolysis of the carboxamide bond in the sulfonylbenzamide moiety to give AE 0001789-descyclopropylamino,

- Desmethylation of the methoxybenzoyl moiety,

- Hydrolytic cleavage of the carboxamide bond in the methoxybenzoyl moiety of:

• The parent compound to form AE 0001789-cyclopropylsulfamoylbenzamide and AE 0001789-anisic acid or of


• AE 0001789-desmethyl to form AE 0001789-cyclopropylsulfamoylbenzamide alone.

Summary of the available dermal penetration study

The in vitro dermal penetration rate of 14C labelled test material (sulfonylbenzamide-ring) in an agrochemical formulation (SC450) following single dermal application to excised human and rat dermatomed skin was determined (M-279026-01-2). The application was performed at two concentrations corresponding to the neat product (nominal value = 225 mg test material/mL formulation) and a representative spray dilution (nominal value = 0.27 mg test material/mL formulation). The two formulations were applied at a rate of 10 µL/cm2 to unoccluded skin samples mounted in flow-through diffusion cells.

Eight hours post-application, the remaining dose material was washed off the skin with freshly prepared 1% v/v Tween 80 in PBS (phosphate buffered saline) using natural sponge swabs. Receptor fluid samples were collected at hourly intervals for the duration of the study (24 hours). The solubility of the test material in the receptor fluid was demonstrated to be sufficient for the study. At the end of the study (24 hours) the skin samples were tape-stripped to remove residual surface dose and the stratum corneum.

For the high dose formulation, the mean percentage of [14C]-radiolabeled test material directly absorbed over a period of 24 hours was <0.005% and 0.06% for the human and rat skin, respectively. The mean percentage of [14C]-radiolabeled test material considered to be potentially absorbable over a period of 24 hours was 0.85% and 1.76% for the human and rat skin, respectively, yielding a factor difference of 2.1 between the two species for the neat product.

For the low dose formulation, the mean percentage total directly absorbed was 0.08% and 0.43% for the human and rat skin, respectively. The mean percentage potentially absorbable over a period of 24 hours was 1.50% and 4.11% for the human and rat skin, respectively, yielding a factor difference of 2.7 between the two species for the spray dilution.

Conclusion and assessment of the toxicokinetic behaviour of the test substance

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), assessment of the toxicokinetic behaviour of the substance is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physicochemical and toxicological properties according to the relevant Guidance (ECHA, 2017) and taking into account the available toxicokinetic studies on the registration substance.

N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide is a mono-constituent substance with a molecular weight of 374.42 g/mol. The substance is a solid powder at 20 °C with a water solubility of 1.09 g/L at 20 °C (pH 6.9) and vapour pressure of 1.0E-5 Pa at 20 °C. The median particle size is 6.7 µm. The log Pow was estimated to be -0.80 at pH 7 and 1.77 at pH 4.

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).

Absorption Oral

The biokinetic behaviour and metabolism of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide in the rat was investigated following a single oral dose to male and female animals, employing a low dose level of 2 mg/kg bw and a high dose of 200 mg/kg bw and using two different label positions with ¹⁴C uniformly labeled in the sulfonylbenzamide and the methoxy benzoyl ring.

The gastro-intestinal absorption started immediately after dosing. The maximum plasma levels were reached already after 10 – 60 minutes. The absorption was complete at the low dose (2 mg/kg b.w.) in both sexes; between 82 % (methoxy benzoyl label) or even more than 90 % (sulfonylbenzamide label) of the dose recovered was renally excreted or remained in the body at sacrifice, 96 hours after dosing (without GIT). In the high dose group, a certain degree of saturation of the absorption process could be observed as shown by the maximum dose-normalized plasma levels. They were lower in the high-dose than in the low-dose animals and the faecal excretion was increased in the high-dose animals by a factor of 2 – 3 compared to the low-dose animals.

Absorption Dermal

For N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide, an in vitro dermal penetration study using human skin is available (M-279026-01-2, 2006). The study was performed by the use of the test substance in an agrochemical formulation (SC450) at two concentrations: the neat product (nominal value = 225 mg/mL) and a representative spray dilution (nominal value = 0.27 mg/mL) were tested. For the neat product, the mean percentage of the test substance considered to be potentially absorbable over a period of 24 h was 0.85%. For the spray dilution, the mean percentage of the test substance potentially absorbable over a period of 24 h was 1.5%.

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 (ECHA, 2017).

The physicochemical properties (log Pow and water solubility) of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide and the molecular weight are in a range suggestive that dermal absorption is possible, however, based on the low log Pow of -0.80, dermal absorption might be limited as its hydrophilicity may hinder crossing the stratum corneum.

If a substance shows skin irritating or corrosive properties, damage to the skin surface may enhance penetration. N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide is not skin irritating/corrosive. Therefore, no enhanced penetration of the substance due to skin damage is expected. 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). Data on skin sensitisation show that the substance is not sensitising, and thus, experimental data do not provide conclusive evidence on possible skin penetration.

Overall, taking all available information into account, and placing most emphasis on the in vitro penetration studies, dermal absorption of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide is considered to be low. Based on the available in vitro data, a dermal absorption rate of 1.5% is considered.

Absorption Inhalation

N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide is a solid powder with very low vapour pressure (1.0E-5 Pa at 20 °C), and therefore low volatility. Under normal use and handling conditions, inhalation exposure and availability for respiratory absorption of the substance in the form of vapours, gases, or mists is considered to be negligible (ECHA, 2017). However, the substance may be available for absorption after inhalation of aerosols, (e.g. spraying of the formulated product). 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. Particles deposited in the nasopharyngeal/thoracic region will mainly be cleared from the airways by the mucocilliary mechanism and swallowed. The particle size distribution of the pure substance shows that the median particle size (L50) was 6.7 µm while the L10 was 1.9 µm. 90% of the particle volume or particle mass had a lower particle diameter than 13.6 µm (L90). This indicates that a significant fraction of the pure powder form of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide has the potential to be inhaled should exposure occur, and will primarily be deposited in the alveolar region of the respiratory tract.

Some minor non-specific treatment-related systemic effects were observed in the acute inhalation toxicity study (key, 2004). However, this gives limited information on the levels of absorption. For the oral route, the available toxicokinetic data indicate high levels of absorption. Therefore, for the purposes of route-to-route extrapolation for DNEL derivation, absorption via the inhalation route is assumed to be equivalent that for the oral route, namely 100%.

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, 2012).

In the available toxicokinetic studies, the distribution of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide -related radioactivity within the body was uniform. The quantity of radioactivity in all organs and tissues was low compared to administered dose and always lower than the level in blood, indicating no tendency of bioaccumulation. A particular concentration of radioactivity in certain organs and tissues was not observed. The highest residue levels were detected in kidney (renal medulla) acting as main organ for excretion. Therefore, it can be concluded that residues of N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide do not accumulate in any of the organs, tissues or glands. As observed in the whole body autoradiography studies particularly residues from glandular organs or tissues responsible for hormonal regulation (such as adrenal, testis, or thyroid gland) were rapidly depleted in parallel with depletion from the other organs and tissues.

Metabolism

N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxybenzamide was metabolised only to a low extent. The parent compound was the main component detected in urine and faeces, accounting in all tests for more than 76% of the dose administered. The proposed metabolic pathway is described above in the summary of the available toxicokinetic studies and a schematic is attached in study summaries of M-276053-01-1 and M-274975-01-1 in the technical dossier.

Excretion

The major routes of excretion for substances from the systemic circulation are the urine and/or the faeces (via bile and directly from the GI mucosa). In the available toxicokinetic studies, the test substance was rapidly excreted, with urinary excretion being the predominant route. Faecal excretion was also observed, but to a lesser extent. No significant expiration of 14C-labelled volatiles was observed.

References

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

ECHA (2012). Guidance on information requirements and chemical safety assessment, Chapter R.8: Characterisation of dose [concentration]-response for human health. Version 2.1, November 2012. European Chemicals Agency, Finland.