1-methyl-2-pyrrolidone

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 July 2000 to 13 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: in vitro dermal skin penetration study with human and rat skin

Version / remarks:

(OECD draft Test Guideline 1999, to meet requirements of Directive 91/414/EEC)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-2-pyrrolidone

EC Number:

212-828-1

EC Name:

1-methyl-2-pyrrolidone

Cas Number:

872-50-4

Molecular formula:

C5H9NO

IUPAC Name:

1-methylpyrrolidin-2-one

Test material form:

liquid

Details on test material:

- Name of test material: Non-labelled NMP
- source: Fisher Scientific, UK
- analytical purity: 99.84 %
- Batch no.: 9757560 367
- Expiry date: Sept. 2002
- Storage: Ambient

- Name of test material: [14C] NMP
- source: NEN Life Science Products, UK
- specific activity: 1.3 mCi/mmol
- radiochemical purity: > 99 %
- Batch no.: 3353-258
- Storage: at < -15°C in the dark

Specific details on test material used for the study:

- Name of test material: Non-labelled NMP
- source: Fisher Scientific, UK
- analytical purity: 99.84 %
- Batch no.: 9757560 367
- Expiry date: Sept. 2002
- Storage: Ambient

- Name of test material: [14C] NMP
- source: NEN Life Science Products, UK
- specific activity: 1.3 mCi/mmol
- radiochemical purity: > 99 %
- Batch no.: 3353-258
- Storage: at < -15°C in the dark

Radiolabelling:

yes

Remarks:

Batch no.: 3353-258

Test animals

Details on test animals or test system and environmental conditions:

TEST SKINS
(all skins supplied by the UK Human Tissue Bank, University College of London, UK)

Full thikness human skin for:
Finite and infinite applications
- Source: human breast skin from 20 and 30 year old female and abdominal skin from a 35 year old female donor
- Storge: -20°C
Skin integrity
- Source: 23 year and 57 year old female donor
- Storge: -20°C
BOTH
- Preparation:
1 thawed to room temperature and any fat removed
2 resulting full thickness skin membrane swabbed with approximately 70% v/v ethanol/water to remove residual fat and blood
3 immediately wiped dry and re-hydrated with a thin film of water prior to dermatoming

Full thikness rat skin
- Source: Male CD Sprague Dawley rats
- Weight at study initiation: 125-148g
- Age at study initiation: 28-32 d
- Supplier: Charles River (UK) Limited, Margate, Kent, UK
- Preparation:
1 rats killed by cervical dislocation following an overdose of isofluorane anaesthetic
2 dorsal region of the rat was shaved with clippers and skin removed
3 connective tissue and any residual fat removed from the dermis carefully
4 resulting full thickness membrane pinned out on a dermatome board (cork board with raised rubber cutting surface)
5 mini-dermatome used to cut slices of skin which contained epidermis and some dermis (approximately 200-400 μm thick)

IN VITRO CELL/ENVIRONMENTAL CONDITIONS
- Method: flow through diffusion cell
- Cell supplier: Crown Glass Company Inc ., Clinton, New Jersey, USA
- Cell diameter: 0.64 cm2 skin exposure area
- receptor fluid pump rate: 2 ml/hr
- recpetor content changes / hour: approx. 8
- Temperature (°C): 32°C usinhg a water-heated manifold
- pH receptor fliud: pH 7.4 (phosphate-buffered physiological saline)

Administration / exposure

Vehicle:

other: limonene (97% purity) or water

Duration of exposure:

3h

Doses:

100 %; 30 % in water and 65 % in limonene

No. of animals per group:

5-7 cells

Control animals:

no

Details on in vitro test system (if applicable):

Dermatome membranes (about 200 - 400 µm thickness) were prepared from human (female, breast skin) and rat (male SD) skin and were mounted in flow-through diffusion cells (Crown Glass Company, USA) maintained at about 32 °C. The receptor fluid was phosphate buffered saline (pH 7.4).
Neat NMP or solutions in limonene (65 % v/v) or water (35 % v/v) were applied using a finite dose volume (6 µL to 0.64 cm² skin) to human and rat skin. Receptor fluid samples were collected at 2 min intervals for the first hour and subsequently at 30 min intervals for up to 3 h after application. Carbon filter traps were placed immediately following administration, which were removed, extracted and analyzed at the end of the study. Skin was swabbed with 1 % (v/v) Tween 80 in distilled water on cotton buds and the swabs extracted and analyzed. The skin was digested and analyzed. For infinite dose application (250 µl to 0.64 cm² skin) to human skin only, the receptor fluid samples were collected at 15 min intervals for the duration of the study to ensure steady-state absorption had been reached. Immediately following administration, the application site was occluded.
Human skin integrity was examined by assessing the permeability coefficient (Kp) of tritiated water ([3H]water) before and after application of NMP. Radioactivity was measured by means of liquid scintillation chromatography (LSC).

Results and discussion

Absorption in different matrices:

see tables under "Any other information on results incl. tables"

Total recovery:

see tables under "Any other information on results incl. tables"

Percutaneous absorption

Percutaneous absorption 1

Dose:

9.73

Parameter:

percentage

Absorption:

ca. 37 %

Remarks on result:

other: 3 hours

Remarks:

undiluted NMP; human skin

Percutaneous absorption 2

Dose:

9.73

Parameter:

percentage

Absorption:

ca. 53 %

Remarks on result:

other: 3 hours

Remarks:

undiluted NMP; rat skin

Percutaneous absorption 3

Dose:

7.34

Parameter:

percentage

Absorption:

ca. 90 %

Remarks on result:

other: 3 hours

Remarks:

65 % NMP in limonene; human skin

Percutaneous absorption 4

Dose:

7.34

Parameter:

percentage

Absorption:

ca. 98 %

Remarks on result:

other: 3 hours

Remarks:

65 % NMP in limonene, rat skin

Conversion factor human vs. animal skin:

1.4

Any other information on results incl. tables

The distribution of radioactivity following a single finite dose application of the three solutions 14C-NMP to human (H) and rat (R) skin are summarized as follows (expressed as mean percent applied dose):

1) Finite dose application:

NMP	100% (undiluted)		30% in water		65% in limonene
	H	R	H	R	H	R
Termination (h)	3	3	3	3	3	3
Dose (mg/cm²)	9.73	9.73	2.77	2.77	7.34	7.34
% absorbed (0-1h)*	7.24	23.32	0.55	0.67	44.36	88.93
% absorbed (0-3h)*	37.34	53.21	20.60	39.19	90.04	97.71
skin %	20.15	15.80	36.69	23.92	1.85	0.39
Total recovery.	96.91	98.69	101.15	100.75	95.13	99.0
Lag time (h)	0.69	0.24	1.98	0.45	0.19	-
Absorption rate (µg/cm²/h)	1650	3113	578.61	905.5	6331	12905

* = in receptor fluid

 

The ratio of the absorption rates for 30% v/v in water : 100% NMP : 65% v/v in limonene were as follows:

Ratio of absorption	30% in water	100% (undiluted)	65% in limonene
Human skin	0.35 :	1.0 :	3.8
Rat skin	0.29 :	1.0 :	4.1

 

2) Infinite dose application:

NMP	100 %	30 % in water	65 % in limonene
Dose (mg/cm²)	399.6	109.5	257.8
lag time (h)	2.59	1.32	1.6 - 3.3
Absorptionrate (µg/cm²/h)	10057	114.2	64957

 

Applicant's summary and conclusion

Executive summary:

Dermatome membranes (about 200 - 400 µm thickness) were prepared from human (female, breast skin) and rat (male SD) skin and were mounted in flow-through diffusion cells (Crown Glass Company, USA) maintained at about 32 °C, the receptor fluid was phosphate buffered saline (pH 7.4). Neat NMP or solutions in limonene (65 % v/v) or water (35 % v/v) were applied using a finite dose volume (6 µL to 0.64 cm² skin) to human and rat skin. Receptor fluid samples were collected at 2 min intervals for the first hour and subsequently at 30 min intervals for up to 3 h after application. Carbon filter traps were placed immediately following administration, which were removed, extracted and analyzed at the end of the study. Skin was swabbed with 1 % (v/v) Tween 80 in distilled water on cotton buds and the swabs extracted and analyzed.

The skin was digested and analyzed. For infinite dose application (250 µL to 0.64 cm² skin) to human skin only, the receptor fluid samples were collected at 15 min intervals for the duration of the study to ensure steady-state absorption had been reached. Immediately following administration, the application site was occluded. Human skin integrity was examined by assessing the permeability coefficient (Kp) of tritiated water ([3H]) before and after application of NMP. Radioactivity was measured by means of liquid scintillation chromatography (LSC).

NMP is rapidly absorbed through human and rat skin within 1 h when applied either as neat or diluted in limonene (65 % v/v). Absorption was most pronounced for the 65 % (v/v) solution in limonene. The absorption for the 30 % (v/v) solution in water was lower than for the neat NMP.

Rat skin overestimated human skin permeability for tested NMP preparations. The difference was particularly apparent in the first hour. The absorption profile for the finite and infinite doses were similar over the first hour but differed thereafter.

The absorption profiles for the finite and infinite doses were very similar over the first hour of exposure. However after this time, there appeared to be different mechanisms affecting the absorption of N-methylpyrrolidone for the two dose regimes . The results therefore highlight the need to discriminate between the finite and infinite dose regimes and its influence on the data produced .

It would appear that N-methylpyrrolidone affects the integrity of skin as function of exposure time and may act as an enhancer of its own absorption within the first hour of absorption .