2-(4-tert-butylbenzyl)propionaldehyde

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

adopted April 13, 2004 (“Skin Absorption: in vitro Method”)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-tert-butylbenzyl)propionaldehyde

EC Number:

201-289-8

EC Name:

2-(4-tert-butylbenzyl)propionaldehyde

Cas Number:

80-54-6

Molecular formula:

C14H20O

IUPAC Name:

3-(4-tert-butylphenyl)-2-methylpropanal

Radiolabelling:

yes

Remarks:

3-14C

Administration / exposure

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: dermatomed human skin preparations; surgically removed skin from abdomen and/or breast
- Donors: age <65 years, without scars and strechmarks.
- Preparative technique: Dermatomized human skin preparations were supplied in suitable pieces frozen. Each skin preparation was hydrated in physiological saline for about 10 minutes before mounting to the diffusion cells which are filled up with physiological saline with protease inhibitor. The prepared diffusion cells were stored overnight in a refrigerator.
- Thickness of skin (in mm): 200 – 400 μm (217 - 389 μm, 261 – 400 μm, 200 – 389 μm, 238 – 386 μm for exp. 1&2, exp. 3&4, exp. 5&6, exp.7&8, respectively),
- Membrane integrity check: only visually intact skin with a TEER (impedance value) above 1 kΩ was used. Electrical resistance (TEER) was tested with a LCR bridge.
- Storage conditions: maximum storage time of 3 days in a refrigerator or 12 months at -20°C.

PRINCIPLES OF ASSAY
- Diffusion cells: Modified Franz cells, exposed skin area: 1 cm²
- Number of cells: 8 cells/group, minimum 4 donors/group
- Exposure time: 24 h
- Receptor fluid: tap water (exp. 1, 3, 5, 7) and tap water with 0.01 % (w/w), NaN3 (exp. 2, 4, 6, 8)
- Solubility of test substance in receptor fluid: The test substance is soluble in tap water with 0.033 g/L. Taking into account the amounts of a.i. administered for the high dose (target dose: 95 μg/cm²) and for the low dose (target dose: 5 μg/cm²), the amounts of test substance penetrated through the skin during the experiments as well as the volume of receptor fluid used (flow: 2.3 mL/h; corresponds to about 55 mL over the exposure period of 24 h), no rate limiting effects on the diffusion process by saturation of the aqueous receptor fluid were present.

- Flow-through system: continuous flow of 2.3 mL/h
- Test temperature: The temperature of the water was adjusted to 36 ± 1°C to realize a surface temperature of the skin preparation of 32 ± 1°C.
- Occlusion: charcoal filter and Fixomull® Stretch adhesive fleece to fix the filters after application (semi-occlusive conditions)
- Formuations: Four test-substance preparations (dose groups) of Lysmeral that reflect commercial applications were tested:
Formulation 1 - ethanol in water 70%
Formulation 2 - Oil in water
Formulation 3 - Water in oil
Formulation 4 - Silicone in oil

- Doses: The test-substance preparations were realized with pure 14C-Lysmeral and had the following target concentrations:
Dose group 1: 1.9 % 14C-Lysmeral in formulation 1
Dose group 2: 0.1 % 14C-Lysmeral in formulation 2
Dose group 3: 0.1 % 14C-Lysmeral in formulation 3
Dose group 4: 0.1 % 14C-Lysmeral in formulation 4
An application volume corresponding to about 5 mg/cm² was administered and lead to the following target doses of 14C-Lysmeral
Dose group 1: 95.0 μg Lysmeral / cm²
Dose group 2: 5.0 μg Lysmeral / cm²
Dose group 3: 5.0 μg Lysmeral / cm²
Dose group 4: 5.0 μg Lysmeral / cm²
Taking the specific activity of 14C-Lysmeral into account, the target doses correspond to the following radioactive target doses:
Dose group 1: 1035.5 kBq / cm²
Dose group 2: 54.5 kBq / cm²
Dose group 3: 54.5 kBq / cm²
Dose group 4: 54.5 kBq / cm²

- Experiments:
Experiment 1: dose group 1 (Formulation 1) 24h sampling, 24h skin wash, terminal procedure at 24h.
Experiment 2: dose group 1 (Formulation 1) 72h sampling, 24h+72h skin wash, terminal procedure at 72h. Methanol extraction of skin preparations.
Experiment 3: dose group 2 (Formulation 2) 24h sampling, 24h skin wash, terminal procedure at 24h.
Experiment 4: dose group 2 (Formulation 2) 72h sampling, 24h+72h skin wash, terminal procedure at 72h. Methanol extraction of skin preparations.
Experiment 5: dose group 3 (Formulation 3) 24h sampling, 24h skin wash, terminal procedure at 24h.
Experiment 6: dose group 3 (Formulation 3) 72h sampling, 24h+72h skin wash, terminal procedure at 72h. Methanol extraction of skin preparations.
Experiment 7: dose group 4 (Formulation 4) 24h sampling, 24h skin wash, terminal procedure at 24h.
Experiment 8: dose group 4 (Formulation 4) 72h sampling, 24h+72h skin wash, terminal procedure at 72h. Methanol extraction of skin preparations.
Experiment 9: dose group 1 (Formulation 1); Control group
Experiment 10: dose group 2 (Formulation 2); Control group

- Controls: Controls were performed to demonstrate the extractability of the test-substance on the skin preparations without exposure time. Therefore, the test-substance preparations were applied on untreated skin preparations and were extracted immediately after application.

- Sampling:
Receptor Fluid:
Experiment 1,3,5,7: pre-dose (collection about 15 minutes), 0-1; 1-2; 2-3; 3-4; 4-5; 5-6; 6-7; 7-8, 8-10; 10-12; 12-14; 14-16; 16-18; 18-21, 21-24 hours after application.
Experiment 2,4,6,8: pre-dose (collection about 15 minutes), 0-1; 1-2; 2-3; 3-4; 4-5; 5-6; 6-7; 7-8, 8-10; 10-12; 12-14; 14-16; 16-18; 18-21, 21-24, 24-48, 48-72 hours after application.
Samples for the mass balance:
Extract of charcoal filter
Post exposure wash: skin surface was thoroughly washed twice with washing fluid (Sodium-laurylethersulfate diluted 1:140 w/w in tap water). The washing fluid, tap water, pipette tips and cotton swabs used were stored for analysis. All parts of the diffusion cell (with the exception of the stainless steel clamp) were extracted in a suitable solvent.
Tape stripping: Twenty tape strips were taken. The tapes were pooled into three samples (the first 2 tapes as sample 1, the subsequent 9 tapes as sample 2 and the last 9 samples as sample 3) for analysis.
Remaining skin:
Experiment 1,3,5,7: The remaining skin was separated into dermis and epidermis by heat separation and was analyzed separately.
Experiment 2,4,6,8,9,10: The remaining skin were extracted immediately after the stripping procedure or application (for controls). Each skin piece was placed into Eppendorf reaction vials and two beads as well 0.8 mL methanol were added and extraction and homogenization was performed in the tissue lyzer following a defined protocol. The homogenates of each skin were centrifuged for 10 minutes at 4500 rpm. The
supernatants were measured by LSC. Aliquots of the non extractable residues in the pellet were measured by LSC after addition of about 5 mL Soluene®-350 and incubation at room temperature for at least 3 days at room temperature.

- Test sample analyses: Liquid Scintillation Counting (LSC) of all study samples; prior digestion by Soluene for skin samples. High Performance liquid chromatography for radiochemical purity of stock preparation of 14C BMHCA and purity/ concentration in formulation vehicles.

Results and discussion

Percutaneous absorption

Percutaneous absorption 1

Time point:

24 h

Dose:

1.9% in Ethanol (70%)

Parameter:

percentage

Absorption:

7.08 %

Remarks on result:

other:

Remarks:

(Absorbed dose) + (Epidermis + Dermis * 80%) = 5.31+((1.5+0.71)*(100-20)%) = 7.08%

Percutaneous absorption 2

Time point:

24 h

Dose:

0.1 % in Oil-in-Water

Parameter:

percentage

Absorption:

5.67 %

Remarks on result:

other:

Remarks:

(Absorbed dose) + (Epidermis + Dermis * 61%) = 4.77+((0.69+0.78)*(100-39)%) = 5.67%

Percutaneous absorption 3

Time point:

24 h

Dose:

0.1 % in Silicone-in-Water

Parameter:

percentage

Absorption:

4.68 %

Remarks on result:

other:

Remarks:

(Absorbed dose) + (Epidermis + Dermis * 74%) = 3.5+((0.96+0.64)*(100-26)%) = 4.68%

Percutaneous absorption 4

Time point:

24 h

Dose:

0.1 % in Water-in-Oil

Parameter:

percentage

Absorption:

5.83 %

Remarks on result:

other:

Remarks:

(Absorbed dose) + (Epidermis + Dermis * 68%) = 4.83+((0.74+0.73)*(100-32)%) = 5.83%

Any other information on results incl. tables

Summary of [14C]-BMHCA penetration through and into human skin after single topical application of target doses of 95.0 μg/cm² and 5.0 μg/cm² of test substance formulated in different test-substance preparations expressed as % of applied doses

%	Dose group		Exp. 1		Exp. 2		Exp. 3		Exp. 4		Exp. 5		Exp. 6		Exp. 7		Exp. 8
	Vehicle		EtOH 70%				Silicone in water				Water in oil				Oil in water
	exposure/sampling		24h/24h	SD	24h/72h	SD	24h/24h	SD	24h/72h	SD	24h/24h	SD	24h/72h	SD	24h/24h	SD	24h/72h	SD
	target concentration	[mg/g]	19,0		19,0		1,0		1,0		1,0		1,0		1,0		1,0
	target dose level of test substance	[µg/cm²]	95,0		95,0		5,0		5,0		5,0		5,0		5,0		5,0
	mean actual nominal dose level test substance	[µg/cm²]	91,3		95,8		4,7		4,2		4,7		5,0		4,7		4,6
Dislodgeable dose	charcoal filter	mean % of applied dose	55,35	2,37	56,49	12,93	55,13	4,15	62,88	4,48	32,87	6,61	28,24	9,99	35,11	4,41	23,92	3,10
	membrane washing after 24 hours	mean % of applied dose	11,60	3,55	13,20	4,00	14,45	6,41	12,28	1,76	53,85	8,85	50,37	13,07	47,75	5,01	56,23	5,43
	membrane washing after 72 hours	mean % of applied dose	-	-	1,80	1,19	-	-	2,78	0,88	-	-	1,21	0,97	-	-	0,82	0,33
	donor chamber washing	mean % of applied dose	1,65	1,46	1,99	1,06	4,03	2,47	2,62	1,85	1,06	0,68	0,96	0,61	1,71	0,90	0,92	0,47
	sum		68,60	3,77	73,48	12,35	73,61	5,57	80,55	4,28	87,77	4,85	84,86	14,82	84,58	4,40	81,89	5,14
Dose associated to tape strips	tape strip sample 1 (first two strips)	mean % of applied dose	0,90	0,33	1,52	1,17	1,19	0,42	0,88	0,46	1,16	0,75	0,62	0,51	1,88	0,77	0,22	0,17
	tape strip sample 2 (3-11)	mean % of applied dose	2,40	1,00	2,42	0,86	2,60	1,38	1,16	0,71	1,84	1,07	0,94	0,79	3,11	1,65	0,32	0,25
	tape strip sample 3 (12-20)	mean % of applied dose	1,02	0,85	0,31	0,24	0,58	0,27	0,12	0,16	0,26	0,13	0,11	0,17	0,41	0,17	0,02	0,03
Dose associated to remaining skin	epidermal membrane	mean % of applied dose	1,50	0,49	-	-	0,96	0,18	-	-	0,74	0,31	-	-	0,69	0,31	-	-
	dermal membrane	mean % of applied dose	0,71	0,28	-	-	0,64	0,23	-	-	0,73	0,35	-	-	0,78	0,17	-	-
	skin residue	mean % of applied dose	-	-	0,32	0,11	-	-	0,25	0,10	-	-	0,24	0,15	-	-	0,18	0,06
	skin extract	mean % of applied dose	-	-	1,31	0,33	-	-	0,71	0,24	-	-	0,50	0,48	-	-	0,28	0,12
Absorbed dose	sum receptor samples 0 - 24 h	mean % of applied dose	1,78	0,65	2,20	0,82	1,62	0,72	2,49	1,56	3,81	2,85	5,49	3,76	3,43	1,68	3,17	1,49
	receptor fluid	mean % of applied dose	0,04	0,02	0,00	0,00	0,03	0,01	0,00	0,00	0,13	0,08	0,00	0,00	0,12	0,03	0,00	0,00
	receptor chamber washing	mean % of applied dose	3,49	1,57	3,09	1,78	1,85	0,72	2,55	1,18	0,89	0,65	2,33	1,70	1,22	0,46	1,80	0,83
	sum		5,31	2,22	5,29	2,52	3,50	1,31	5,04	2,60	4,83	3,54	7,82	5,42	4,77	2,16	4,97	2,26
Total recovery		mean % of applied dose	80,44	1,83	84,67	13,80	83,08	3,28	88,72	2,97	97,32	3,91	91,01	13,82	96,21	2,98	87,88	3,44