N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Purity not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Scott R C and Clowes H M (1992). In vitro Percutaneous Absorption Experiments: A Guide to the Techniques for Use in Toxicology Assessments. Toxicology Methods, Vol. 2, No. 2, pp.113-123.

Principles of method if other than guideline:

The in vitro absorption of various dyes been measured to obtain information on their potential to be absorbed through human and pig epidermis.
Absorption was measured using glass diffusion cells employing established methodology (Scott and Clowes, 1992).

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

255-473-8

EC Name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

41642-51-7

Molecular formula:

C20H19N7O3

IUPAC Name:

N-[2-[(2,6-dicyano-4-nitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Test material form:

solid

Specific details on test material used for the study:

No further details specified in the study report.

Radiolabelling:

no

Test animals

Species:

other: Human and pig epidermis

Sex:

not specified

Details on test animals or test system and environmental conditions:

Extraneous tissue was removed from human whole skin samples and pig whole skin was separated from the cartilage of pig ears. The whole skin samples from both species were immersed in water at 60 °C for 40-50 seconds. The epidermis was gently teased off the dermis and stored deep frozen on aluminium foil until required for use.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: 0.5% TWEEN 80 in distilled water

Duration of exposure:

55 hours

Doses:

The application rate was 200 μl cm-2 (equivalent to 200 μg dye cm-2).

No. of animals per group:

6 human epidermis and 6 pig epidermis per test substance.

Control animals:

no

Details on in vitro test system (if applicable):

Samples of human or pig epidermis were mounted in glass diffusion cells and the integrity of the membranes determined by measurement of their permeability to tritiated water. Membranes displaying a permeability coefficient of <1.5 x 10-3cm hr-1 (human) or <4.5 x 10-3cm hr-1 (pig) were regarded as being undamaged and used for exposure to the test dyes.
Each dye was mixed with a solution of 0.5% TWEEN 80 in distilled water to give a dye concentration of 1000 μg ml-1 and at this concentration all the dyes remained in suspension. The suspensions were ultra-sonicated to disperse any large particles and were applied to the epidermal membranes immediately after preparation to ensure maximum homogeneity. The application rate was 200 μl cm-2 (equivalent to 200 μg dye cm-2). The donor chambers were occluded to prevent any evaporation of the vehicle during the exposure period (55hr). At recorded intervals throughout the exposure period, samples (0.5 ml) of the receptor fluid (50% ethanol in distilled water) were taken from the receptor chamber for analysis. The volume of fluid in the receptor chamber was maintained by the addition of 0.5 ml of fresh receptor fluid to the chamber immediately after the removal of each sample.
The samples taken during the exposure period were analysed by high performance liquid chromatography (HPLC), with the limits of determination varying between 0.02 and 0.05 μg ml-1, dependent upon the dye. The results of the analyses were used to calculate the amount (μg cm-2) of dye absorbed at each sample time point and to determine the absorption profiles (μg cm-2 v time). The absorption rates (μg cm-2 hr-1) were calculated from the slope of the profiles between a chosen range of time points. The maximum absorption rates is represented by the linear portion of the profile.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

The most poorly absorbed of the dyes was Disperse Blue 165, from which no absorption was detected through human epidermis (<0.004 μg cm-2 hr-1) and from pig epidermis absorption was only detected 48 hr after exposure through 3 of the 6 membranes used (0.013 μg cm·2 hr-1).

Total recovery:

Not examined

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

55 h

Dose:

200 μl cm-2

Parameter:

rate

Absorption:

0 mg cm-2 h-1

Remarks on result:

other: Human epidermis

Percutaneous absorption 2

Key result

Time point:

55 h

Dose:

200 μl cm-2

Parameter:

rate

Absorption:

0 mg cm-2 h-1

Remarks on result:

other: Pig epidermis

Conversion factor human vs. animal skin:

For all 6 dyes absorption rates were faster through pig epidermis than through human epidermis. With the exception of Disperse Blue 165, where the difference between pig and human epidermis could only be calculated as >3.25, the factors of difference (pig > human).

Any other information on results incl. tables

SUMMARY OF THE MEAN ABSORPTION RATES THROUGH HUMAN AND PIG EPIDERMIS

Dye	Mean Absorption rates (n = 6)
	Human Epidermis		Pig Epidermis
	Time Period (hr)	μg cm^-2hr^-1 = SEM	Time Period (hr)	μg cm^-2hr^-1 = SEM
Disperse Blue 165	1 – 10 1 – 55	<0.007 <0.001	1 – 10 31 – 55	< 0.007 0.013 ± 0.006

Where absorption was below the limit of determination LOD, the value for the LOD has been included to calculate the mean.

Applicant's summary and conclusion

Conclusions:

The Disperse Blue 165 was slowly absorbed (maximum rates <0.004 cm-2 hr-1 for human and 0.013 μg cm-2 hr-1 for pig).

Executive summary:

The in vitro absorption of disperse dyes (Disperse Blue 165) has been measured through human and pig epidermis. The dyes were prepared as suspensions (1000 μg ml^-1) in a 0.5% solution of TWEEN 80 in distilled water which were applied to the epidermal membranes at a rate of 200 μl cm^-2 (= 200 μg cm^-2). Absorption was measured under occlusion during an exposure period of 55 hr.

 

The most slowly absorbed dye was Disperse Blue 165 (maximum rates <0.004 μg cm^-2 hr^-1 for human and 0.013 μg cm^-2 hr^-1 for pig).