Amines, N-C12–14 (even numbered)-alkyltrimethylenedi-, reaction products with chloroacetic acid and diethylenetriamine, N-C12–14 (even numbered)-alkyl derivs.

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:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-C12–C14(even numbered)-alkyltrimethylenedi-, reaction products with chloroacetic acid

Molecular formula:

n.a. (UVCB substance)

IUPAC Name:

Amines, N-C12–C14(even numbered)-alkyltrimethylenedi-, reaction products with chloroacetic acid

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid - liquid: aqueous solution

Details on test material:

- Name of test material: DOPA-Glycinate

Radiolabelling:

yes

Test animals

Species:

human

Administration / exposure

Type of coverage:

open

Vehicle:

other: tap water

Duration of exposure:

6 hours
Termination by washing skin sample surface with commercial soap concentrate followed by rinsing with a dilute soap solution and drying with tissue swabs.

Doses:

Test preparation 1: 14C DOPA-Glycinate (21.094 % a.i.) determined by radioactivity
Test preparation 2: 14C DOPA-Glycinate (0.228 % a.i.) determined by radioactivity (diluted with tap water)

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Skin samples: Split-thickness human skin membrane samples

APPARATUS
Automated flow-through diffusion cell apparatus (Scott/Dick, University of Newcastle-upon-Tyne, UK), placed in a steel manifold heated via a circulating water bath to maintain the skin surface temperature. The cells were connected to multi-channel peristaltic pumps from their afferent ports with the receptor fluid effluent dropping via fine bore tubing into scintillation vials on a fraction collector.

PRINCIPLES OF ASSAY
- Volume applied: 10 µL/cm²; the surface area of exposed skin within the cells was 0.64 cm².
- Receptor fluid: Tissue culture medium containing bovine serum albumin (ca 5%, w/v), glucose (ca 1%, w/v), streptomycin (0.1 mg/mL) and penicillin G (100 units/mL). The receptor chamber volume was 0.25 mL, the peristaltic pumps were adjusted to maintain a flow-rate of ca 1.5 mL/h.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

no effects

Total recovery:

Test preparation 1, 20 % (w/w): 101.69 % (SD = 3.04%)
Test preparation 2, 0.2 % (w/w): 97.10 % (SD = 2.99%)

Percutaneous absorption

Percutaneous absorption 1

Time point:

24 h

Dose:

20%

Parameter:

percentage

Remarks:

potentially absorbable dose (sum of the absorbed dose, exposed skin and stratum corneum tape strips 3–20)

Absorption:

1.1 %

Percutaneous absorption 2

Time point:

24 h

Dose:

0.2%

Parameter:

percentage

Remarks:

potentially absorbable dose (sum of the absorbed dose, exposed skin and stratum corneum tape strips 3–20)

Absorption:

15.1 %

Percutaneous absorption 3

Time point:

24 h

Dose:

20%

Parameter:

percentage

Remarks:

Dermal delivery (exposed skin + absorbed dose)

Absorption:

0.6 %

Percutaneous absorption 4

Time point:

24 h

Dose:

0.2%

Parameter:

percentage

Remarks:

Dermal delivery (exposed skin + absorbed dose)

Absorption:

5 %

Any other information on results incl. tables

Test preparation 1, 20 % (w/w):99.76 % of the applied dose was removed by washing at 6 h post application. At 24 h post application, the total dislodgeable dose was 100.35 % of the applied dose. The stratum corneum retained 0.72 % of the applied dose, 0.37 % was removed with the first 5 tape strips.

Absorbed dose: 0.01 % (0.18 ng equivalent/cm²)

Dermal delivery: 0.62 % (13.17 ng equivalent /cm²)

Potentially absorbable dose: 0.97 % (20.53 ng equivalent /cm²)

Test preparation 2, 0.2 % (w/w):73.66 % of the applied dose was removed by washing at 6 h post application. At 24 h post application, the total dislodgeable dose was 75.30 % of the applied dose. The stratum corneum retained 16.08 % of the applied dose, 9.71 % was removed with the first 5 tape strips.

Absorbed dose: 0.11 % (0.02 ng equivalent /cm²)

Dermal delivery: 5.04 % (1.01 ng equivalent /cm²)

Potentially absorbable dose: 11.42 % (2.28 ng equivalent /cm²)

 

 

Table A6.2-4:Details on human skin.

Donor no.	Sex/age of donor	Site of sampling	Supplier	Membrane full-thickness (µm)	Membrane split-thickness (µm)
0164	F/28Y	Breast	St. Johns Hospital	1010–1150	380–390
0161	F/36Y	Abdomen	BUPA	1020–1080	390–400
0162	F/35Y	Abdomen	BUPA	700–1190	390–400
0179	F/84Y	Breast	Nottingham	1130	400
0169	F/82Y	Breast	Nottingham	1000	400
0180	F/74Y	Breast	Nottingham	940–1000	400
0166	F/33Y	Abdomen	Transkin	1240	400

 

 

Table A6.2-5:Summary of the results (mean values).

Test preparation	1		2
Target concentration of active substance	20 % (w/w)		0.2 % (w/w)
Active substance concentration in test preparation by radioactivity	21.016 % (w/w)		0.199 % (w/w)
Application rate of Test preparation	10.06 mg/cm²		10 µl/cm²
Application rate of test item	2114 µg equivalent/cm²		19.94 µg equivalent/cm²
Distribution	% applied dose	ng equivalent /cm²	% applied dose	ng equivalent /cm²
Dislodgeable dose 6 h (skin wash+tissue swab+pipette tips)	99.76	2108.47	73.66	14.69
Total dislodgeable dose (dislodgeable dose 6 h +stratum corneum + unexposed skin + cell wash)	100.35	2120.83	75.30	15.02
Unabsorbed dose	101.07	2136.17	92.06	18.36
Absorbed dose (cumulative receptor fluid + receptor rinse)	0.01	0.18	0.11	0.02
Dermal delivery (exposed skin + absorbed dose)	0.62	13.17	5.04	1.01
Potentially absorbable dose (dermal delivery + stratum corneum tape strips)	0.97	20.53	11.42	2.28
Mass balance (unabsorbed dose + dermal delivery)	101.69	2149.34	97.10	19.36

 

According to EFSA Guidance on Dermal Absorption (2012) as well as the EU Guidance Document

on Dermal Absorption (2004) only the first 2 tape strips should be discarded. On that basis dermal absorption for both low and high doses have been re-calculated.  

 

Recalculation disregarding 2 rather than 5 tape strips:

 

Prep 1 (20% a.i.)
	Cell 1	Cell 2	Cell 3	Cell 4	Cell 8	Cell 9	Cell 10	Cell 12	Cell 13	Cell 14	Mean	STDEV
1	0.008	0.019	0.08	0.068	0.18		0.282	0.12	0.023	0.144	0.103	0.090
2	0.003	0.011	0.141	0.099	0.093		0.295	0.063	0.01	0.201	0.102	0.098
Stratum total	0.04	0.1	0.99	0.69	0.95		1.58	0.62	0.16	1.33	0.718	0.549
minus 1+2	0.029	0.07	0.769	0.523	0.677		1.003	0.437	0.127	0.985	0.513	0.378
Dermal delivery	0.02	0.09	0.77	0.27	1.35		1.73	0.82	0.15	0.41	0.623	0.597
Absorbable Dose	0.049	0.16	1.539	0.793	2.027		2.733	1.257	0.277	1.395	1.137	0.907
Prep 2 (0.2% a.i.)
	Cell 16	Cell 17	Cell 18	Cell 19	Cell 24	Cell 25	Cell 26	Cell 28	Cell 29	Cell 30	Mean	STDEV
1	3.11	2.24	2.84	4.42	3.36	3.89	4.82	3.78	4.87	6.06	3.939	1.131
2	1.66	1.8	1.71	2.33	1.73	2.53	2.19	2.26	2.5	2.26	2.097	0.338
Stratum total	12.02	13.59	14.46	19.48	15.39	15.49	20.79	16.73	14.73	18.17	16.085	2.718
minus 1+2	7.25	9.55	9.91	12.73	10.3	9.07	13.78	10.69	7.36	9.85	10.049	2.052
Dermal delivery	2.56	2.52	3.84	3.15	8.21	9.37	6.12	7.47	3.15	4.02	5.041	2.538
Absorbable Dose	9.81	12.07	13.75	15.88	18.51	18.44	19.9	18.16	10.51	13.87	15.090	3.611

Applicant's summary and conclusion

Conclusions:

The systemically available dose of DOPA-Glycinate (= dermal delivery: exposed skin + absorbed dose) was 0.6±0.6% for the high exposure scenario (20% a.i.), and 5.0±2.5% for the low exposure scenario (0.2% a.i.), respectively.
The potentially absorbable dose of DOPA-Glycinate was 1.1±0.9% for the high exposure scenario (20% a.i.), and 15.1±3.6% for the low exposure scenario (0.2% a.i.), respectively.

Executive summary:

The percutaneous absorption of [¹⁴C]-DOPA-Glycinate was tested according to OECD 428: Skin Absorption: In Vitro Method (2004).

Split-thickness human skin membranes were mounted into flow-through diffusion cells. Receptor fluid, consisting of bovine serum albumin (ca. 5%), glucose (ca 1%), streptomycin (0.1 mg/mL), and penicillin G (100 U/mL), was pumped underneath the skin at a flow rate of ca 1.5 mL/h. Receptor fluid was collected in hourly fractions and analysed by liquid scintillation counting.

Test preparation 1, 20 % (w/w): Mass balance 101.69 % (SD = 3.04%), thus considered to be complete.

Absorbed dose: 0.01 % (0.18 ng equivalent/cm²)

Dermal delivery: 0.62 % (13.17 ng equivalent /cm²)

Potentially absorbable dose(sum of absorbed dose, exposed skin and stratum corneum tape strips 6–20): 0.97 % (20.53 ng equivalent /cm²)

Test preparation 2, 0.2 % (w/w): Mass balance 97.10 % (SD = 2.99%), thus considered to be complete.

Absorbed dose: 0.11 % (0.02 ng equivalent /cm²)

Dermal delivery: 5.04 % (1.01 ng equivalent /cm²)

Potentially absorbable dose (sum of absorbed dose, exposed skin and stratum corneum tape strips 6–20): 11.42 % (2.28 ng equivalent /cm²)

 

However, according to EFSA Guidance on Dermal Absorption (2012) as well as the EU Guidance Document on Dermal Absorption (2004) only the first 2 tape strips should be discarded. On that basis the potentially absorbable dose for both low and high doses has been re-calculated.  

The potentially absorbable dose (the sum of the absorbed dose, exposed skin and stratum corneum tape strips 3–20 may be used for risk assessment; this corresponds to 1.1 ±0.9% for the high exposure (20% a.i.), and 15.1±3.6% for the low exposure scenario (0.2 % a.i.), respectively.

For REACH-purposes the

"Dermal absorption represents the amount of topically applied test substance that is found in the epidermis (stratum corneum excluded) and in the dermis, and this quantity is therefore taken as systemically available. Dermal absorption is influenced by many factors, e.g. physico-chemical properties of the substance, its vehicle and concentration, and the exposure pattern (e.g. occlusion of the application site) as well as the skin site of the body (for review see ECETOC, 1993; Howes et al, 1996; Schaefer and Redelmaier, 1996). The term percutaneous penetration refers to in vitro experiments and represents the amount of topically applied test substance that is found in the receptor fluid – this quantity is taken as systemically available. " (Guidance on Information requirements and chemical safety assessment, R.7c).