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

Dermal absorption

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-320-2

EC Name:

-

Cas Number:

221667-31-8

Molecular formula:

C18H18N205S

IUPAC Name:

N-[4-(cyclopropylcarbamoyl)benzenesulfonyl]-2-methoxybenzamide

Radiolabelling:

yes

Remarks:

14C labelled test substance (Sulfonylbenzamide-ring)

Administration / exposure

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".

Results and discussion

Total recovery:

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

Percutaneous absorption

Percutaneous absorption 1

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

225 mg/mL

Parameter:

percentage

Absorption:

0.85 %

Remarks on result:

other: human skin

Percutaneous absorption 2

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.27 mg/mL

Parameter:

percentage

Absorption:

1.5 %

Remarks on result:

other: human skin

Percutaneous absorption 3

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

225 mg/mL

Parameter:

percentage

Absorption:

1.76 %

Remarks on result:

other: rat skin

Percutaneous absorption 4

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.