Registration Dossier

Toxicological information

Dermal absorption

Currently viewing:

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2008-09-23 to 2009-03-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to guideline, well documented

Data source

Reference
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Qualifier:
according to
Guideline:
other: Environmental Health and Safety Publications, Series on Testing and Assessment no. 28. Guidance document for the conduct of skin absorption studies (Paris, March 2004).
Qualifier:
according to
Guideline:
other: Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients. SCCP/0970/06, updated March 2006.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Chemical name : Toluene-2,5-diamine sulphateCAS Reg no. : 615-50-9COLIPA number : A005Molecular formula : C7H10N2.H2O4SMolecular weight : 220.25 g.mol-1 (free base 122.17 g.mol-1)Solubility in water : 10 weight % in water at pH 7.Stability in solution : stable in water and water/acetone (4:1 v/v) for 8 days at ambient temperature in the absence of light.Batch number : 2346Sample number : R99053665Aspect : white-grey powder
Radiolabelling:
yes

Test animals

Species:
human
Sex:
female
Details on test animals and environmental conditions:
Human skin membranes were prepared from frozen skin samples, present at TNO Quality of Life. Human skin (derived from abdomen and/or breast) was obtained from six female donors, directly after surgery. The skin from donor 4 was obtained from the back region instead of derived from abdomen and/or breast. In each experiment, skin obtained from three different donors was used.Donor 1: TNA14/08, born in 1979, arrival at TNO on 5 June 2008 (groups A/E)Donor 2: TNA15/08, born in 1954, arrival at TNO on 6 June 2008 (groups A/E)Donor 3: TNA16/08, born in 1975, arrival at TNO on 13 June 2008 (groups D/H)Donor 4: TNA23/08, born in 1956, arrival at TNO on 11 July 2008 (groups B/F, C/G)Donor 5: TNA26/08, born in 1951, arrival at TNO on 18 July 2008 (groups A/E, B/F, C/G, D/H)Donor 6: TNA28/08, born in 1954, arrival at TNO on 22 August 2008 (groups B/F, C/G, D/H)The transportation of the skin to the laboratory was carried out as soon as possible after dissection, while the skin was place in a plastic container that was kept on ice. Subcutaneous fat was removed and the skin was stored in aluminium foil at <−18 °C until use. The skin of donor 1 was stored overnight at 2-10 °C before subcutaneous fat was removed. Informed consent was provided by all donors.

Administration / exposure

Type of coverage:
not specified
Vehicle:
not specified
Duration of exposure:
30 ± 5 minutes
Doses:
From 47.9 ± 0.8 to 1599.1 ± 285.6 μg.cm-2The test substance was tested at four concentrations (i.e. 0.25 %, 0.8 %, 2.4 % and 7.2 % (w/w)) in two formulations. One formulation was used undiluted and the second one was mixed 1:1 with Welloxon Perfect (it contains 6 % hydrogenperoxide) immediately prior to the experiment.
No. of animals per group:
Six skin membranes from three donors in each test group
Control animals:
no
Remarks:
in vitro study
Details on study design:
Toluene-2,5-diamine sulphate was applied topically to the skin membranes according to the design below. The exposure time was 30 ± 5 minutes and receptor fluid samples were collected from 0 – 24 hours following application.
Details on in vitro test system (if applicable):
The study was split in five separate experiments (group D, donor three was repeated once due to a dosing/application error in experiment four), each in which twoformulations were evaluated (except D, donor 3). The dose preparations were prepared on the day of application. The radiochemical purity of the tracer in the dosepreparations was verified by radio-HPLC.For groups E, F, G and H, the formulations were mixed 1:1 (w/w) with Welloxon Perfect 6% prior to application.The dose preparations were applied using a disposable plastic rod (except dose solution A which was applied using a positive displacement pipette) and spread evenly on the skin surface within the donor compartment (ca 20 mg.cm-2). The concentration and homogeneity of [14C]Toluene-2,5-diamine sulphate in the dose preparations were checked by randomly taking 6 weighed aliquots from each formulation. Aliquots were dissolved using tetrahydrofuran (THF) and measured by LSC. For homogeneity, a coefficient of variation lower than 15 % was considered sufficient.After 24 h of application, the mass balance of the test substance was determined from the following samples: receptor fluid samples, skin wash, receptor compartment wash, donor compartment wash, tape strips, and digested skin.

Results and discussion

Absorption in different matrices:
TThe mean total absorption, defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash and the skin membranes (excluding tape strips) was 1.75 μg.cm-2 (3.65 % of the applied dose, A) and 2.09 μg.cm-2 (3.49 %, E), 16.27 μg.cm-2 (9.55 %, B) and 6.92 μg.cm-2 (3.88 %, F), 47.23 μg.cm-2 (9.25 %, C) and 21.81 μg.cm-2 (3.90 %, G) and 101.03 μg.cm-2 (6.43 %, D) and 54.03 μg.cm-2 (3.49 %, H).
Total recovery:
The mean recovery of [14C]Toluene-2,5-diamine sulphate ranged from 99.18 ± 3.21 % to 102.80 ± 5.05 % of the applied dose
Percutaneous absorptionopen allclose all
Dose:
0.25 % PTD
Parameter:
percentage
Absorption:
3.23 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
Non oxidative conditions (group A)
Dose:
0.25% PTD
Parameter:
percentage
Absorption:
2.15 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
oxidative conditions (group E)
Dose:
0.8 % PTD
Parameter:
percentage
Absorption:
8.27 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
non-oxidative conditions (group B)
Dose:
0.8 % PTD
Parameter:
percentage
Absorption:
2.64 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
oxidative conditions (group F)
Dose:
2.4 % PTD
Parameter:
percentage
Absorption:
8.17 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
non-oxidative conditions (group C)
Dose:
2.4 % PTD
Parameter:
percentage
Absorption:
2.83 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
oxidative conditions (group G)
Dose:
7.2 % PTD
Parameter:
percentage
Absorption:
5.77 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
non-oxidative conditions (group D)
Dose:
7.2 % PTD
Parameter:
percentage
Absorption:
2.76 %
Remarks on result:
other: 0.5 to 2 hours
Remarks:
oxidative conditions (group H)

Any other information on results incl. tables

Mean absorption of [14C]PTD into the receptor fluid and total absorption under non-oxidative and oxidative conditions (from formulations containing 0.25 %, 0.8 %, 2.4 % and 7.2 % PTD)

 Applied dose (μg.cm-2)  Group  Absorbed dose (μg.cm-2)  Total absorption (μg.cm-2)  Absorbed dose (% of applied dose) Total absorption (% of applied dose) 
 Non-oxidative conditions          
 47.9 1.55 ± 0.47  1.75 ± 0.52  3.23 ± 0.98  3.65 ± 1.08
173.4  B  14.09 ± 2.78  16.27 ± 2.94  8.27 ± 2.07  9.55 ± 2.17
 514.2  C  41.87 ± 11.66  47.23 ± 13.35  8.17 ± 2.15  9.25 ± 2.54
 1559.2  D  90.82 ± 33.76  101.03 ± 36.67  5.77 ± 2.20  6.43 ± 2.36
 Oxidative conditions          
 60.9  E  1.29 ± 0.16  2.09 ± 0.53  2.15 ± 0.51  3.49 ± 1.02
 181.5  F  4.61 ± 1.36  6.92 ± 1.56  2.64 ± 0.91  3.88 ± 0.92
 566.9  G  15.96 ± 4.52  21.81 ± 4.87  2.83 ± 0.79  3.90 ± 0.93
 1574.4  H  42.63 ± 11.23  54.03 ± 15.38  2.76 ± 0.75  3.49 ± 0.92

Applicant's summary and conclusion

Conclusions:
The mean total absorption, defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash and the skin membranes (excluding tape strips) was 1.75 μg.cm-2 (3.65 % of the applied dose, A) and 2.09 μg.cm-2 (3.49 %, E), 16.27 μg.cm-2 (9.55 %, B) and 6.92 μg.cm-2 (3.88 %, F), 47.23 μg.cm-2 (9.25 %, C) and 21.81 μg.cm-2 (3.90 %, G) and 101.03 μg.cm-2 (6.43 %, D) and 54.03 μg.cm-2 (3.49 %, H).The mean recovery of [14C]Toluene-2,5-diamine sulphate ranged from 99.18 ± 3.21 % to 102.80 ± 5.05 % of the applied dose
Executive summary:

The percutaneous absorption of [14C]Toluene-2,5-diamine sulphate (PTD) was evaluated on human (split-thickness) skin membranes. Eighteen skin membranes from three different human skin donors were used. The exposure time was 30 ± 5 minutes, with 23.5 hours post-exposure time.

Both under non-oxidative and oxidative conditions (i.e. the conditions of human exposure), a linear increase in the amount of PTD in the receptor fluid and the total amount absorbed was observed. The mean absorption into the receptor fluid tended to slightly decrease at higher applied doses although this appears not statistically significant. Furthermore, under oxidative conditions, the amount of radio-activity recovered from the tape-strips (i.e. stratum corneum) was considerably higher when compared to non-oxidative conditions.

The cumulative absorption profiles for [14C]Toluene-2,5-diamine sulphate were comparable between groups with most of the absorption taking place within the first four hours after application.

The mean total absorption, defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash and the skin membranes (excluding tape strips) was 1.75 μg.cm-2 (3.65 % of the applied dose, A) and 2.09 μg.cm-2 (3.49 %, E),

16.27 μg.cm-2 (9.55 %, B) and 6.92 μg.cm-2 (3.88 %, F), 47.23 μg.cm-2 (9.25 %, C) and 21.81 μg.cm-2 (3.90 %, G) and 101.03 μg.cm-2 (6.43 %, D) and 54.03 μg.cm-2 (3.49 %, H).