4,4'-methylenedianiline

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Principles of method if other than guideline:

Investigation of percutaneous absorption of 4.4'-methylenedianiline (MDA) after topical application to full-thickness skin from male Fischer 344 rats in flow-through diffusion cells, on four separate occasions.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenedianiline

EC Number:

202-974-4

EC Name:

4,4'-methylenedianiline

Cas Number:

101-77-9

Molecular formula:

C13H14N2

IUPAC Name:

4,4'-methylenedianiline

Specific details on test material used for the study:

RADIOLABELLING INFORMATION
- Radiochemical purity: >95%
- Specific activity: 56 mCi/mmol
- Locations of the label: [ring14C]4,4 '-methylenedianiline (Amersham)

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-OLAC (Oxford, Oxon, UK)
- Diet: CRM pellets, Biosure (Manea, Cambs., UK); ad libitum
- Water: ad libitum

Administration / exposure

Type of coverage:

other: occlusive and open

Vehicle:

ethanol

Duration of exposure:

72 h

Doses:

- Actual doses: 39.4, 20.1, 21.6, 23.6 µg/cm²
- Dose volume: 5-10 µL

No. of animals per group:

4

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: dorsal region of male Fischer 344 rats
- Type of skin: full thickness
- Preparative technique: following sacrificion, the dorsal region was shaved with animal clippers and the shaved area was cut out with dissecting scissors. The rat skin sections were placed, epidermal side uppermost, on a plastic dissecting board and circles, 1.7 cm in diameter, were cut out using a circular sharpened steel cutter. Excess subcutaneous tissue was removed with a scalpel.

PRINCIPLES OF ASSAY
- Flow-through diffusion cell system: consisted of seven teflon diffusion cells and a fraction collector (Crown Glass Co Inc., Somerville, NJ, USA), a thermostatically controlled Churchill water circulator and a model 202V/AA16 cassette peristaltic pump (Watson-Marlow, Falmouth, Cornwall, UK). The receptor volume of each diffusion cell was 130 ul.
- Receptor fluid: HEPES buffered Hanks' balanced salt solution supplemented with 0.5% (v/v) gentamicin, continuously flowing
- Test temperature: 32°C
- the skin was placed in the diffusion cells in the flow-through apparatus, and allowed to equilibrate for about 30min.
- aliquots of 5-10 µL [14C]-MDA (17.7-40.6 µg/cm²) in ethanol were applied to the exposed skin surface.
- occlusion with teflon caps or left open.
- receptor fluid (1.5 mL) was collected hourly for up to 72h.
- at the end of the experiment (72h) the epidermal surface of the skin was washed, and the washings and skin were counted to determine radioactivity remaining on the skin surface.

ANALYSIS
- Liquid scintillation counting

STATISTICAL EVALUATION:
- Student`s t-Test for unpaired data, level of significance p<0.05.

Results and discussion

Absorption in different matrices:

See table 1

Total recovery:

- Total recovery: 94.5 +/-6 %

Percutaneous absorption

Percutaneous absorption 1

Dose:

20.1-39.4 µg/cm³

Parameter:

percentage

Absorption:

ca. 6.1 %

Remarks on result:

other: 72 h

Remarks:

open

Percutaneous absorption 2

Dose:

20.1-39.4 µg/cm²

Parameter:

percentage

Absorption:

ca. 13.3 %

Remarks on result:

other: 72h

Remarks:

occlusive

Any other information on results incl. tables

Table 1: Recovery of radioactivity at 72h after topical application of [14C] MDA to rat skin in vitro (% of applied dose).

	Rat (unoccluded) (n=12)	Rat (occluded) (n=16)
Receptor fluid	6.1 ± 2	13.3 ± 4.0*
Skin surface	25.5 ± 6.8	21.5 ± 7.2
Diffusion cell	5.4 ± 4.4	3.9 ± 2.6
Teflon cap	-	0.3 ± 0.2
Skin surface	57 ± 5.6	53.1 ± 8.6
Total	94.5 ± 6.0	92.1 ± 6.5

* significantly different from unoccluded value (p<0.05)

A typical absorption profile showed a lag phase of about 2 hr before a rapid increase in absorption, reaching an equilibrium at 72 hr (data not shown).