Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2012 to 02 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Principles of method if other than guideline:

The study was also performed according to the additional cosmetic guidelines listed below, with no deviations:
- Cosmetic Ingredients: Guidelines for Percutaneous Absorption/Penetration, The European Cosmetic Toiletry and Perfumery Association COLIPA, 1997.
- SCCP/0970/06 Opinion: Basic Criteria for the in vitro assessment of dermal absorption of cosmetic ingredients, adopted by the SCCNFP during the 7th plenary meeting of 28 March 2006.
- SCCP/Notes of guidance for the testing of cosmetic ingredients and their safety evaluation, 6th revision, December 19, 2006.
- SCCP/Special meeting on dermal absorption/NoS: Meeting Report, Special meeting on dermal issues, October 7, 2008.
- SCCS/Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients, June 22, 2010.

GLP compliance:

no

Remarks:

GLP like study following international guidelines

Test material

Test material information

Constituent 1

Reference substance name:

Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

EC Number:

800-172-4

Cas Number:

398141-87-2

Molecular formula:

Complex UVCB substance

IUPAC Name:

Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Test material form:

other: liquid

Details on test material:

- Storage condition of test material: At room temperature.
- Analytical purity: 100%
- Expiration date of the lot/batch: 25 August 2012

Radiolabelling:

no

Test animals

Species:

human

Sex:

not specified

Administration / exposure

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Duration of exposure:

24 hours

Doses:

- Nominal doses: 10.3 mg test material per cm², theoretical value.
- Actual doses: The actual dose for each application was not calculated, however a reference value for each chamber was determined and used for calculating the mass balance and the percentage of dermal delivery.
- Reference value calculated as follows: A 10 µL aliquot of the test material was pipette into a 20 mL flask and left un-covered for 24 hours. The pipette tips were washed with 2 mL of acetonitrile. The concentration of these solutions was used as the reference value.
- Dose volume: 10 µL.
- Rationale for dose selection: According to the guidelines cited the application of the test material to the skin should mimic realistic conditions. Thus the 10 µL dose volume used corresponds to the realistic in use conditions.

No. of animals per group:

The skin from three donors was used.

Control animals:

no

Details on study design:

APPLICATION OF DOSE: The test material was pipetted directly onto the skin in the respective donor chamber. After which the pipette tips were washed in 2 mL of acetonitrile.

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Skin samples were washed nine times with 1 mL of acetonitrile (extraction solution). Washing solutions were collected and combined in a 20 mL flask, which was taken to volume by adding additional extraction solution.
- Time after start of exposure: 24 hours.

ANALYSIS
- Method type(s) for identification: LC-MS/MS.
- Conditions: Pre column, 0.5 µ x 0.004 ID; Column, 2.6 µ, 50 x 4.6 mm; Flow, 200 µL/min; Injection volume, 20 µL; Column oven, 50 ºC; Time, 13.0 min.
- Solutions:
> Eluent A: 0.1 % formic acid in H2O/acetonitrile (95:5, v/v).
> Eluent B: 0.1 % formic acid in H2O/acetonitrile (5:95, v/v).
- Detection Method: Electrospray ionisation.
- Limits of detection and quantification: The limit of quantitation is 6.02 ng/mL in receptor and extraction solutions. The limit of detection is 5.00 ng/mL.
- Sample storage: Collected samples were stored in tightly closed vessels at 2-8 ºC until analysis, if not analysed immediately.
- Calibration and quality control samples: Samples were prepared by diluting the test material in acetonitrile, yielding samples at concentrations ranging from 2.01 to 1254 ng/mL. Samples were stored at approximately 4ºC in a refrigerator.
- Samples preparation with internal standard: 7-Ethoxycoumarin (99.7% purity) diluted to 1073 µg/mL in methanol and phosphate buffer was added for further dilutions. Samples included calibration, quality control and test samples. Samples were stored at approximately 4 ºC in a refrigerator.

STRIPPING
- Method: After the incubation period the exterior regions of the isolated skin pieces were removed using scissors and extracted with 2 mL extraction solution. Then the stratum corneum was stripped twice using a transparent film. The film pieces were collected in a tube filled with 2 mL extraction solution and submitted for LC-MS/MS analysis to determine the amount of the test material adsorbed and extracted for analysis.

SEPARATION OF SKIN MEMBRANES
- Method: Isolated skin pieces were wrapped in aluminum foil, covered with an approximately 140 g weight and placed on a heating plate for 30 seconds at 55°C. Then the remaining epidermis was gently peeled off the dermis using forceps. All skin compartments were extracted separately for analysis.

SKIN EXTRACTION
- Method: At the end of the separation procedure reference item that had penetrated into the skin was quantified by extraction with 2 mL extraction solution. The extraction was performed sealed at room temperature overnight or for at least 8 hours. The skin extracts were stored at -20°C, if not analysed immediately. The test material was analysed as the relevant compound.
- Control: As a control for all experiments, one skin sample was extracted without prior treatment with the test items in order to determine a possible influence on the analytics of the skin itself. This skin sample was not used in parallel in a diffusion chamber or treated like the skin samples in the experiment, but stripped and the remaining skin extracted in the same way as the samples.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human skin was supplied dermatomized by biopredic, Rennes, France.
- Type of skin: Human skin free from adipose tissue was used from the abdomen of three different donors.
- Thickness of skin (in µm): 400 – 600 µm.
- Membrane integrity check: Conductivity across the skin was determined by measuring the impedance (resistance to alternating current) before and after treatment (at 24 hours, after samples were washed). Impedance was measured by filling the donor chambers with receptor fluid. All diffusion chambers are equipped with platinum electrodes connecting to a conductometer. After the impedance was measured the receptor solution was removed from the donor chamber and depleted. Before application the skin was dried using cellulose. The integrity was checked in each diffusion cell with the exception of number 5, where no conductivity value could be determined after the monitoring period. A slight but not significant increase in conductivity was observed for all other diffusion cells.

PRINCIPLES OF ASSAY
- Diffusion cell: A total of six glass diffusion cells were used, with a diameter of 1.135 cm. Cells were divided into the donor and receptor chambers.
- Receptor fluid: 20% ethanol/PBS (20:80, v/v).
- Extraction solution: Acetonitrile.
- Solubility of test substance in receptor fluid and extraction solution: Approximately 480 µg/mL.
- Flow-through system: The receptor fluid was pumped through each receptor chamber by a 6-channel peristaltic pump, at a flow rate of 0.8 to 1.1 mL/h.
- Test temperature: Receptor chamber temperature 32 ± 1.0 ºC.
- Occlusion: Non-occluded conditions.
- Sampling:
> Sampling intervals: Receptor vessels were replaced and sampled at the following time points; 0, 0.5, 2, 4, 8, 12, 16, 20, 23 and 24 hours.
> The samples collected for the first hour were checked (pre run), but were not reported, since there is a delay time of about 1 hour caused by the length of the tubes from the receptor chambers to the receptor vials. Then the receptor vials were changed at the time points listed above. After 24 hours the content of the receptor vials reflects 23 hours. Additionally the tubes and the remaining volume in the chamber itself were depleted in a second fraction (24 hours).
> Storage: Collected eluates were weighed and stored at -20 ºC until analysis could be performed.

CONTROLS
Positive control: Benzoic acid; last performed in May 2012 yielding a mean dermal absorption of 93.7% ± 5.73%.
Negative control: 2-Ethylhexyl trans-4-methoxycinnamate; last performed in November 2011 yielding a mean dermal absorption of 0.476% ± 0.294%.
Controls: Performed at least once a year by the testing laboratory to confirm the sensitivity of the skin penetration system.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

- Receptor fluid: 0.0458 ± 0.0179% at 24 hours.
- Wash solution and the supernatant of impedance measurement after 24 hours: 89.1 ± 18.2%.
- Skin preparation: Epidermis 0.227 ± 0.1%, Dermis 0.571 ± 0.138%.
- Stratum corneum: 0.33 ± 0.24%.

Total recovery:

- Total recovery: Total percentage recovery ranged from 76.6% to 113.7%, see table 3.
- Recovery of applied dose acceptable: Four out of the six chambers meet the acceptance criteria. Chamber numbers 3 and 6 were removed from the calculation because the determined mass balance was ≤ 85 %. The remaining chambers still contained skin from all three donors.
- Limit of detection (LOD): 5.00 ng/mL.
- Quantification of values below LOQ: 6.02 ng/mL.

Percutaneous absorption

Dose:

100%

Parameter:

percentage

Absorption:

0.844 %

Remarks on result:

other: 24 hours

Remarks:

± 0.228% of the applied dose (111 ± 29.5 µg/cm²)

Any other information on results incl. tables

Integrity of the Skin

The integrity of the skin was demonstrated prior to application and after the last sampling. The conductivity prior to the experiment was in the acceptable range of < 900 µS/cm for all skin samples used.

Table 2. Compartmental Distribution of the Test Material

Amount of Test Material in	Test Material
	Expressed as µg/cm² of skin surface mean ± SD (n=4)			Expressed as % of applied dose mean ± SD (n=4)
Amount applied	13170	±	11712	100	±	1.26
Wash Solution + SN	201	±	2283	89.1	±	18.2
Receptor Fluid	Cumulative as µg/cm²
0.5	2.08	±	1.51	n.d
2	0.691	±	0.451	n.d
4	0.804	±	0.542	n.d
8	1.01	±	0.670	n.d
12	1.24	±	0.802	n.d
16	1.42	±	0.924	n.d
20	1.58	±	1.02	n.d
23	1.65	±	1.07	n.d
24	8.27	±	6.02	n.d
0 - 24	6.06	±	2.48	0.0458	±	0.0179
Stratum corneum	43.3	±	31.5	0.33	±	0.24
Epidermis	29.9	±	13.1	0.227	±	0.10
Dermis	75.2	±	18.1	0.571	±	0.138
Recovery	13385	±	1557	101.7	±	12.0
Bioavailable portion	111	±	29.5	0.844	±	0.288

Only valid results were used, i.e. those yielding a recovery > 85%.

SN = the supernatant of impedance measurement after 24 hours.

n.d = not determined

 

Table 3. Summary of Results

	Chamber Number
	1	2	3 *	4	5	6 *
Amount of test material applied (µg/cm²)^A	13454	13166	13302	13008	13050	13256
Total amount of test material measured (µg)	13314	14201	10194	14792	11236	10516
Recovery (%)	99.0	107.9	76.6	113.7	86.1	79.3
Total absorption of the test material (µg/cm²)^B	114	100	100	150	80.2	64.5
Total absorption (%)^C	0.844	0.762	0.753	1.16	0.614	0.486

* Invalid results where the recovery was ≤ 85%

A = amount of test material present in 10 µL without the pipetting washing solution (PWL).

B = the value is the sum of the amount of the test material measured in the receptor solution and in the skin extract (epidermis and dermis) of each diffusion cell.

C = percent total absorption = (total amount of the test material penetrated/absorbed(µg/cm²) *100)/ amount of applied test material(µg).

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test material was detected in all samples relevant for dermal delivery, e.g. in the skin extracts as well as in all receptor solution samples showing constant penetration over the sampling time of 24 hours. A higher penetration rate was observed at the beginning (0.5 hours) of the experiment, after which a relatively constant penetration rate was observed until termination.

The quantity of the test material that was absorbed was determined to be 111 ± 29.5 µg/cm² and the quantity not absorbed was 13231 ± 1512 µg/cm². Thus the dermal delivery was determined to be 111 ± 29.5 µg/cm² (0.844 ± 0.228% of the applied dose), where the dermal delivery is the sum of the applied dose found in the treated skin and the receptor fluid at termination. Accordingly the test material was considered to penetrate into viable skin at a very low rate.

Executive summary:

The dermal absorption of the test material was assessed for its potential to permeate human skin in an in vitro dermal delivery study using human skin. In total 3 different skin donors were exposed to 10 µL of the test material in duplicate for 24 hours under non-occluded conditions. Aliquots of the test material were applied directly to the skin of the 6 replicate test chambers and then removed by washing each skin sample nine times with 1 mL extraction solution at termination. PBS/20% EtOH (v/v) was used as the receptor fluid.The samples were analysed by LC-MS/MS for the presence of the test material. The conductivity across the skin samples of each chamber was measured before treatment and after the sampling. No abrupt change in conductivity, indicating a loss of barrier properties of the skin, occurred in any chamber up to termination of the experiment at 24 hours.

 

Four out of the six chambers meet the acceptance criteria, chambers of 3 and 6 were excluded because of their low recovery rates. Therefore 4 chambers were used for the analysis. The stratum corneum was stripped from each of the skin pieces and afterwards the epidermis and dermis were separated. The amount of the test material present in each compartment was determined.

 

Under the conditions of the test, the test material was detected in all samples relevant for dermal delivery, e.g. in the skin extracts as well as in all receptor solution samples showing constant penetration over the sampling time of 24 hours. A higher penetration rate was observed at the beginning (0.5 hours) of the experiment, after which a relatively constant penetration rate was observed until termination. The quantity of the test material that was absorbed was determined to be 111 ± 29.5µg/cm² and the quantity not absorbed was 13231 ± 1512µg/cm². Thus the dermal delivery was determined to be 111 ± 29.5 µg/cm² (0.844 ± 0.228% of the applied dose), where the dermal delivery is the sum of the applied dose found in the treated skin and the receptor fluid at termination. Accordingly the test material was considered to penetrate into viable skin at a very low rate.