1-methyl-2-pyrrolidone

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

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

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

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)

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).

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"

Dose:

9.73

Parameter:

percentage

Absorption:

ca. 37 %

Remarks on result:

other: 3 hours

Remarks:

undiluted NMP; human skin

Dose:

9.73

Parameter:

percentage

Absorption:

ca. 53 %

Remarks on result:

other: 3 hours

Remarks:

undiluted NMP; rat skin

Dose:

7.34

Parameter:

percentage

Absorption:

ca. 90 %

Remarks on result:

other: 3 hours

Remarks:

65 % NMP in limonene; human skin

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

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

 

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 .

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2002 to 27 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Draft Guideline 428 for skin absorption: In vitro method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Specific details on test material used for the study:

- Name of test material: Unlabelled NMP
- source: Sigma Aldrich
- analytical purity: > 99 % (HPLC)
- expiry date: 26 May 2004
- batch: U02681

- Name of test material: [14C] NMP
- source: sponsor (NMP Producers Group)
- specific activity: 33.870 KBq/mg
- radiochemical purity: 98.8 % (HPLC)

Radiolabelling:

yes

Remarks:

by Sponsor (NMP Producers Group)

Species:

human

Strain:

other: Ethnicity: Caucasian

Sex:

female

Details on test animals or test system and environmental conditions:

SKIN SAMPLES
- Additional information: see Table 1
- diffusion cell with a dose exposure area of 0.64 cm2
- receptor fluid for test material used with 10 mM phosphate buffered saline, pH 7.4
- Preparation:
- frozen skin samples were thawed to room temperature
- any fat was carefully removed with a scalpel and the skin examined
- skin was attached to a dermatome cutting board (a cork board with a raised rubber cutting surface), slices of skin cut using a mini-dermatome
- acceptable thickness of skin slices approximately 200-400 µm (confirmed using a Micrometer)
- visual inspection, integrity testing by measuring electrical resistance (fail: less than 20 kΩ/cm2)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 2

Type of coverage:

other: occluded with a cap

Vehicle:

other: distilled water or artificial sweat according to AATCC Test method 15-1997 obtained from Sigma Aldrich, UK

Remarks:

Artificial Sweat: NaCI: 10.0g, Lactic Acid: 1.0g, Na2HPO4: 1.0g, 1-Histidine Monohydrate Hydrochloride: 0.25g

Duration of exposure:

single application, exposure for up to 24 hours

Doses:

- 0.1, 0.3, 1.0, 3.0 10.0 and 30 % NMP in water
- 0.3 and 3.0 % in artificial sweat

No. of animals per group:

not applicable

Control animals:

no

Details on study design:

TEST MATERIAL
- Amount(s) applied: 0.25 mL per cell

Details on in vitro test system (if applicable):

Dermatomed human skin membranes were prepared and fitted inside flow through diffusion cells maintained at 32°C.
Aliquots of radiolabelled test material aqueous solutions (6 in water and 2 in artificial sweat) were dosed to the skin membranes at 250 µL per cell (infinite dose).
Receptor fluid (phosphate buffered saline) passed under the membranes was collected at timed intervals up to 24 hours post dosing. The fractions of receptor fluid collected were assayed for total radioactivity by liquid scintillation counting to enable the absorption rate of the radiolabelled test material to be calculated.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

A linear correlation was evident between the concentration of NMP in the aqueous dose solutions and the absorption rate at both selected time periods with an R2 of 0.98 (0-3 hours) and an R2 of 0.99 (3-7 hours). A summary of the calculated absorption rates is given in table 2.
There were no significant statistical differences between the absorption rates of NMP from 3.0 % and 0.3 % NMP solutions in distilled water and artificial sweat.

Time point:

7 h

Dose:

30 % in water

Parameter:

rate

Absorption:

ca. 0.08 mg cm-2 h-1

Remarks on result:

other: Time point described for 3-7h.

Conversion factor human vs. animal skin:

not applicable

Table 2: The table presents a summary of the calculated absorption rates measurements over two time points. Results are expressed as mean +/- Standard Deviation (SD) Group NMP Concentration (% in water) Absorption Rate* Absorption rate* 0 - 3 h (SD) 3 - 7 h (SD) 1 30.0 36.41 (±42.37) 80.41 (±70.29) 2 10.0 16.90 (±12.20) 35.70 (±19.46) 3 3.0 3.51 (±3.20) 8.57 (±6.21) 4 1.0 1.33 (±0.65) 2.94 (±1.25) 5 0.3 0.36 (±0.27) 1.02 (±0.79) 6 0.1 0.14 (±0.08) 0.32 (±0.13) * = µg/cm^2/hour

Executive summary:

The study was performed to evaluate the absorption rate of the radiolabelled NMP through human skin membranes from a series of aqueous and artificial sweat solutions.

Dermatomed human skin membranes were prepared and fitted inside flow through diffusion cells maintained at 32°C. Aliquots of radiolabelled test material aqueous solutions (6 in water and 2 in artificial sweat) were dosed to the skin membranes at 250 µL per cell (infinite dose). Receptor fluid (phosphate buffered saline) passed under the membranes was collected at timed intervals up to 24 hours post dosing. The fractions of receptor fluid collected were assayed for total radioactivity by liquid scintillation counting (LSC) to enable the absorption rate of the radiolabelled test material to be calculated.

A linear correlation was evident between the concentration of NMP in the aqueous dose solutions and the absorption rate at both selected time periods with an R2 of 0.98 (0-3 hours) and an R2 of 0.99 (3-7 hours).

There were no significant statistical differences between the absorption rates of NMP from 3.0 % and 0.3 % NMP solutions in distilled water and artificial sweat.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 1997 to 8 April 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

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 (as cited in study report): [2-14C] NMP
- source: NEN Life Science Product, USA
- specific activity: 1.61 mCi/mmol
- radiochemical purity: 98.3 %
- Batch no.: 3322-147-2
- Storage: at -20°C in the dark

Radiolabelling:

yes

Remarks:

Batch no.: 322-147-2

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 35-49 days
- Weight at study initiation: 235 - 251 g
- Fasting period before study: not indicated
- Acclimatisation: at least five days before study initiation, health status assessed twice daily
- Housing:
- during accommodation: 5/cage, stainless steel
- during the study: individual metabolism cages, equipped for separate collection of urine and faeces and for collection of expired air
- Diet: ad libitum, Laboratory Animal Diet No. 1 SQC (Special Diets Services, Witham, Essex)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C (19-25°C)
- Humidity: 55 % (40- 70 %)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 28 November 1997 To: 8 April 1998

Type of coverage:

semiocclusive

Vehicle:

other: water or Limonene; purity: 97 %

Duration of exposure:

up to 24 hours (1, 3, 6 and 24 hours)

Doses:

- Actual doses calculated as follows:
- 3, 10, 30, 65 % diluted in water (corresponds to 0.3,1.0, 3.0 and 6.5 mg/cm2)
- 3, 10, 30, 65 % diluted in limonene; 100 % neat NMP undiluted (corresponds to 0.3,1.0, 3.0, 6.5 mg/cm2 and 10 mg/cm2 for the neat NMP)
- Calculation: total dose applied (120 μl over a 12 cm2 area per rat) minus the residual dose on the dose spreader
- Dose volume: 120 µl

No. of animals per group:

Four animals per group, one group for each concentration (=nine groups)

Control animals:

no

Details on study design:

see below

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

- Skin wash: 1.8, 2.3, 1.9, 1.6 and 2.2 % for dose solutions of 3, 10, 30, 65 % in water and 100 % after 24 h
- Skin test site: 2.5, 2.6, 1.3, 0.7 and 0.3 % for dose solutions of 3, 10, 30, 65 % in water and 100 % after 24 h
- Skin carcass, untreated site: 0.1, 2.6, 0.2, 0.4 and 1.2 % for dose solutions of 3, 10, 30, 65 % in water and 100 % after 24 h
- Urine, faeces & cage wash: 0.6, 1.3, 3.5, 14.2 and 30.3 % for dose solutions of 3, 10, 30, 65 % in water and 100 % after 24 h
- Expired air: <0.1, < 0.1, <0.1, 0.1 and 0.2 % for dose solutions of 3, 10, 30, 65 % in water and 100 % after 24 h
- Carbon filters: 94.4, 91.2, 95.9, 83.4 and 67.7 % for dose solutions of 3, 10, 30, 65 % in water and 100 % after 24 h

- Skin wash: 7.4, 5.1, 2.0 and 1.2 % for dose solutions of 3, 10, 30 and 65 % in limonene after 24 h
- Skin test site: 0.8, 0.4, 0.4 and 0.6 % for dose solutions of 3, 10, 30 and 65 % in limonene after 24 h
- Skin carcass, untreated site: 0.5, 1.2, 1.7 and 5.0 % for dose solutions of 3, 10, 30 and 65 % in limonene after 24 h
- Urine, faeces & cage wash: 15.5, 54.5, 70.4 and 37.1 % for dose solutions of 3, 10, 30 and 65 % in limonene after 24 h
- Expired air: 0.1, 0.3, 0.4 and 5.0 % for dose solutions of 3, 10, 30 and 65 % in limonene after 24 h
- Carbon filters: 76.3, 37.2, 26.5 and 55.9 % for dose solutions of 3, 10, 30 and 65 % in limonene after 24 h

Total recovery:

- Total recovery: total recovery ranged from 96.1 % to 106.1 % in the respective groups at each sampling time point (for 24h: 97.5 - 102.8%)
- Recovery of applied dose acceptable: yes
- Results adjusted for incomplete recovery of the applied dose: no
- Limit of detection (LOD): depending on test material, ranged from 0.004 (skin) to 2.66 µg test material/g sample (whole blood & red blood cells)
- data of less the LOD is reported as not detected (ND)

Time point:

24 h

Dose:

100 % NMP

Parameter:

percentage

Absorption:

ca. 32 %

Time point:

24 h

Dose:

65 % NMP in aqueous solution

Parameter:

percentage

Absorption:

ca. 15 %

Time point:

24 h

Dose:

65% NMP in limonene solution

Parameter:

percentage

Absorption:

ca. 42 %

Conversion factor human vs. animal skin:

not relevant

The majority of dose was unabsorbed (> 67 %) and was recovered in the carbon filters above dose site following a single topical application of 100% of NMP.

Only a small proportion of dose remained in the treated skin was washed from the skin, falling from approx. 6 % to < 1 % of dose from 1 to 24 hours.

The radioactivity in the blood, plasma and red blood cells at 1, 3 and 6 hours were all of similar magnitude, approx. 110 µg equivalents per g. The concentration of radioactivity in the liver and kidneys at 6 hours were also similar, 100 and 120 µg equivalents/g, respectively. At 24 hours, the levels of blood/plasma/red blood cells had fallen to approx. 2 µg equivalents/g.

At 24 hours after dosing, 31.6 % of the dose had been absorbed and transported away from the dose site, of which 29.9 % was excreted in the urine (plus cage wash), 0.5 % in the faeces and 0.2 % as expired dioxide.

Conclusions:

A single topical application of 100% N-methylpyrrolidone to the clipped dorsal area of the male rat resulted in approximately one third of the dose being absorbed and two thirds being evaporated from the dose site. Very little dose remained at the dose site (treated skin and skin swabs) at 1 hour after dosing, indicating that the equilibrium between absorption and evaporation of dose was rapidly achieved. The proportion of dose that was absorbed was almost entirely eliminated in the urine by 24 hours after dosing.

Dilution of N-methylpyrrolidone with water decreased the absorption of dose with increasing proportions of water in an almost linear relationship. Again, very little dose remained at the dose site (treated skin and skin swabs) at 1 hour after dosing, indicating that the equilibrium between absorption and evaporation of the N-methylpyrrolidone was rapidly achieved. The smaller proportions of dose that were absorbed were almost entirely eliminated in the urine by 24 hours after dosing.

Dilution of N-methylpyrrolidone with (R)-{+)-limonene from 65:35 to 10:90 N-methylpyrrolidone to (R)-(+)-limonene (% by vol) increased the proportion (% dosed radioactivity) absorbed compared to 100% N-methylpyrrolidone, although the absolute mass (mg) of N-methylpyrrolidone absorbed did not increase. Only when the ratio of N-methylpyrrolidone to (R)-(+)-limonene was 3 : 97 (% by vol) was less(% dosed radioactivity) absorbed. Again, very little dose remained at the dose site (treated skin and skin swabs) at 1 hour after dosing indicating that the equilibrium between absorption and evaporation of the
N-methylpyrrolidone was rapidly achieved. The proportion of dose that was absorbed was almost entirely eliminated in the urine by 24 hours after dosing.

Executive summary:

The absorption of 14C-radiolabelled NMP in male Sprague-Dawley CD rats was determined following a single topical application of undiluted NMP and also diluted at four concentrations (3, 10, 30 and 65 %) in two dose vehicles (water and limonene). For each dose solution, one group of rats was killed at 1, 3, 6 and 24 hours after dosing.

A single topical application of 100 % NMP to the clipped dorsal area of the male rats resulted in approximately one third of the dose being absorbed and two thirds being evaporated from the dose site.

Dilution of NMP with water decreased the absorption of dose with increasing proportions of water in an almost linear relationship.

Dilution of NMP with limonene from 65 : 35 to 10 : 90 increased the proportion (% of radioactivity) absorbed compared to 100 % NMP although the mass (mg) of NMP absorbed did not increase.

Under all conditions, very little dose remained at the dose site at 1 hour after dosing, indicating that the equilibrium between absorption and evaporation of dose was rapidly achieved. The proportion of dose that was absorbed was almost entirely eliminated in the urine by 24 hours after dosing.

Reference 4

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 November 2002 to 26 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

Draft guideline, 2002

Deviations:

yes

Remarks:

Small/No faeces samples for some animals forced the laboratory staff to combine gastrointestinal tracts samples with corresponding faeces samples.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material: Non-radiolabelled N-Methylpyrrolidone
- Source and batch No.of test material: U06354 and Sigma Aldrich, Dorset, UK

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark


SOURCE OF TEST MATERIAL
- Name of test material: labelled [14C]-N-Methylpyrrolid
- Source and lot No.of test material: Safepharm Laboratories Ltd, UK; 1695/001
- Purity test date: 06 June 2002

RADIOLABELLING INFORMATION
- Radiochemical purity: > 97% (HPLC)
- Specific activity: 33.87 kBq/mg
- Locations of the label: C2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. -20 °C in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: radiodilution with non-radiolabelled N-Methylpyrrolidone
- Preliminary purification step: specific activity value of 4 .49 kBq/mg (0.12 µCi/mg)

Radiolabelling:

yes

Remarks:

Batch 1695/001

Species:

rat

Strain:

other: Crl: CD (SD) IGS BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 251-328 g
- Housing: housed in polycarbonate cages in groups of 1-2/cage; single housing during study
- Individual metabolism cages: yes
- Diet: ad libitum; SDS Rat and Mouse Maintenance Diet No. 1, Special Diets Services, 1 Stepfield, Witham, Essex
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 46 - 49
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 November 2002 To: 19 December 2002

Type of coverage:

other: - % coverage: either fully occluded with aluminium foil (group 1); semi-occluded with a carbon filter fitted ca. 0.5 cm above skin (group 2); semi-occluded with a carbon filter fitted ca. 2 cm above skin (group 3); unoccluded (group 4)

Vehicle:

unchanged (no vehicle)

Duration of exposure:

1 hour

Doses:

100 µL/animal over 10cm² skin area

No. of animals per group:

three/group

Control animals:

no

Details on study design:

Twelve male rats each received single dermal application of [14C]-N-Methylpyrrolidone at a target dose level of 100 µL applied over a 10 cm2 area of skin. Approximately 24 h prior to dose application, the fur was clipped from the back of each animal and ca 1 h prior to dose application, a rubber O-ring of internal area ca 10 cm2 was glued onto the back of each animal using acrylic glue. The dose site was occluded as follows:

Group 1 (3 rats): Fully occluded with aluminum foil
Group 2 (3 rats): Semi-occluded with a carbon filter fitted ca 0.5 cm above the skin
Group 3 (3 rats): Semi-occluded with a carbon filter fitted ca 2 cm above the skin
Group 4 (3 rats): Unoccluded

Following dose application, animals were placed singly in Jencon’s metabowls. At 1 h post dose, the dose site was washed and the animals humanely killed. Excreta, selected tissues, whole blood and plasma samples were obtained and the levels of total radioactivity determined. The levels of total radioactivity present in the occlusion device, jacket and skin wash extract were also determined.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

The dose was rapidly absorbed (within 1 hour of dosing) in each treatment group. Highest absorption was observed in the group where there was no occlusion of the dose site (68.9 %) and lowest absorption in the group where the dose site was fully occluded. A proportion of the dose was removed from the skin by washing the dose site at 1 h post dose. Further absorption of the applied dose is predicted following application of the dose for periods beyond 1 h. A portion of the dose was shown to be volatile and condensed on the full occlusion apparatus or was absorbed into the carbon filters of the semiocclusion device. Radioactivity in the semi-occlusive carbon filters placed 2 cm from dose site was lower than in the filters placed 0.5 cm from the dose site. Some condensation of volatilized radioactivity on the protective jacket in the non-occluded dose site group was observed, but overall levels of unabsorbed radioactivity were lower when compared to other groups.
Selected mean data are given in table 1.

Distribution in tissues
At 1 h post dose the majority of the absorbed dose remained in the carcass (43.4 %), gastrointestinal tract (6.4 %) and tissues (liver: 3.5 %, G.I. tract: 6.4 %) in the non-occluded exposure experiment.

Excretion
In the non-occluded exposure experiments, the levels of total radioactivity in excreta samples accounted for means of 1.2, 1.9, and 0.15 % of administered dose in urine, cage wash and faeces, respectively.

Time point:

1 h

Dose:

0.1 mL

Parameter:

percentage

Absorption:

> 50 %

Remarks on result:

other: in each group of animals

Time point:

1 h

Dose:

0.1 mL

Parameter:

percentage

Absorption:

>= 57 - <= 77 %

Remarks on result:

other: non-occluded group

Time point:

1 h

Dose:

0.1 mL

Parameter:

percentage

Absorption:

>= 45 - <= 58 %

Remarks on result:

other: fully occluded group

Conversion factor human vs. animal skin:

not applicable

Table 1: Selected mean data of absorption for the different experiments after 1 h exposure of NMP with a dose of 100 µL over a 10 cm2 area of skin.

 

Component	% applied			Dose
	Fully Occluded	Semi-occluded (0.5cm)	Semi-occluded (2cm)		Unoccluded
Dosed Skin	8.4	10.9	8.5 37.4 10.5 0.4		8.9 43.4 13.3 3.3
Carcass	31.3	34.9
Other Tissues	9.9	10.8
Excreta	0.8	0.9
Absorbed Dose	50.4	57.5	56.8		68.9
Skin Wash	35.7	16.3	23.9		17.9 NA 4.5
Occlusion Device	7.4	15.6	10.2
Jacket	0.0	1.1	0.0
Unabsorbed Dose	43.2	33.0	34.2		22.4
Total Recovery	93.6	90.5	90.9		91.3

NA=not applicable (dose area not occluded)

Conclusions:

The dose was rapidly absorbed (within 1 hour of dosing) in each treatment group. Highest absorption was observed in the group where there was no occlusion of the dose site and lowest absorption in the group where the dose site was fully occluded .

A proportion of the dose was removed from the skin by washing the dose site at 1 h post dose. Further absorption of the applied dose is predicted following application of the dose for periods beyond 1 h.
A portion of the dose was shown to be volatile and condensed on the full occlusion apparatus or was absorbed into the carbon filters of the semiocclusion device. Radioactivity in the semi-occlusive carbon filters placed 2 cm from dose site was lower than in the filters placed 0 .5 cm from the dose site.
Some condensation of volatilised radioactivity on the protective jacket in the non-occluded dose site group was observed, but overall levels of unabsorbed radioactivity were lower when compared to other groups .

At 1 h post dose the majority of the absorbed dose remained in the carcass, gastrointestinal tract and tissues.

Executive summary:

The study has been designed to determine the distribution of total radioactivity, over a period of 1 h after dermal application of [14C]-N-Methylpyrrolidone, with different degrees of occlusion of the dose application site.

Twelve male rats each received single dermal application of [14C]-N-Methylpyrrolidone at a target dose level of 100 µL applied over a 10 cm2 area of skin. Approximately 24 h prior to dose application, the fur was clipped from the back of each animal and ca 1 h prior to dose application, a rubber O-ring of internal area ca 10 cm2 was glued onto the back of each animal using acrylic glue.

The dose site was occluded as follows:

Group 1 (3 rats): Fully occluded with aluminum foil

Group 2 (3 rats): Semi-occluded with a carbon filter fitted ca 0.5 cm above the skin

Group 3 (3 rats): Semi-occluded with a carbon filter fitted ca 2 cm above the skin

Group 4 (3 rats): Unoccluded

Following dose application, animals were placed singly in Jencon’s metabowls.

At 1 h post dose, the dose site was washed and the animals humanely killed. Excreta, selected tissues, whole blood and plasma samples were obtained and the levels of total radioactivity determined. The levels of total radioactivity present in the occlusion device, jacket and skin wash extract were also determined.

The dose was rapidly absorbed (within 1 hour of dosing) in each treatment group. Highest absorption was observed in the group where there was no occlusion of the dose site and lowest absorption in the group where the dose site was fully occluded. A proportion of the dose was removed from the skin by washing the dose site at 1 h post dose. Further absorption of the applied dose is predicted following application of the dose for periods beyond 1 h. A portion of the dose was shown to be volatile and condensed on the full occlusion apparatus or was absorbed into the carbon filters of the semiocclusion device. Radioactivity in the semi-occlusive carbon filters placed 2 cm from dose site was lower than in the filters placed 0.5 cm from the dose site. Some condensation of volatilized radioactivity on the protective jacket in the non-occluded dose site group was observed, but overall levels of unabsorbed radioactivity were lower when compared to other groups.

At 1 h post dose the majority of the absorbed dose remained in the carcass, gastrointestinal tract and tissues.

Following dermal application of 14C-NMP at a target dose of 0.1 mL/10 cm2 the majority (45-77 %) of the dose in each dose group was rapidly absorbed within 1 hour of dosing.

Highest absorption (57-77 %) was observed in the group where there was no occlusion of the dose site and lowest absorption (45-58 %) in the group where the dose was fully occluded.

Description of key information

Dermal absorption has been extensively studied as it typically poses the greatest potential for human exposure.

Due to its irritant properties, neat NMP is unlikely to remain in voluntary contact with the skin for more than several hours. Dermal penetration through human skin has been shown to be very rapid and the absorption rate is in the range of 1 - 2 mg/cm2/h. These values are 2- to 3-fold lower than those observed in the rat during the same period. Prolonged exposures to neat NMP was shown to increase the permeability of the skin. Skin penetration can also be affected by solvents and to a lesser extent by the state of occlusion. Water inhibits dermal absorption while other organic solvents (e.g., d-limonene) can increase it. The dermal penetration of 10 % NMP in water is 100-fold lower than that of neat NMP, while dilution of NMP with d-limonene can increase the absorption of NMP by as much as 10-fold. The dermal absorption of neat NMP under different conditions indicated that dermal absorption 1 hour post-exposure was greatest under unoccluded conditions (69 %), followed by semi-occluded (57 %) and occluded (50 %) conditions.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

60

Additional information

In vitro studies (animal skin models)

Investigations on dermatomed rat skin in vitro with application of neat NMP or solutions in limonene (65 %) or water (30 %) for up to three hours resulted in absorption rates of 3.113 mg/cm²/h, 12.905 mg/cm²/h and 0.906 mg/cm²/h, respectively. After three hours skin penetration amounted to 53 %, 98 % and 39 % for neat NMP, 65 % NMP in limonene and 30 % NMP in water, respectively (Huntingdon Life Sci., 2002).

In vitro studies (human skin models)

Investigations on dermatomed human skin in vitro with application of neat NMP or solutions in limonene (65 %) or water (30 %) for up to three hours resulted in absorption rates of 1.650 mg/cm²/h, 6.331 mg/cm²/h and 0.579 mg/cm²/h, respectively. After three hours skin penetration amounted to 37 %, 90 % and 21 % for neat NMP, 65 % NMP in limonene and 30 % NMP in water, respectively. Examinations of the effect of NMP on skin integrity showed the ability of NMP to enhance its own absorption (Huntingdon Life Sci., 2002).

Another study showed no differences between absorption rates of NMP tested as 3.0 % or 0.3 % solutions in distilled water and artificial sweat (SafePharm Laboratories Ltd., 2004).

A comparative skin penetration study with split human skin on different commercial solvents showed a permeation rate of NMP of 17.1 mg/cm²/h, similar to that of DMSO (Ursin et al., 1995). The skin penetration of NMP through human skin is 1 - 2 mg/cm²/h (Huntingdon Life Sci., 2002).

Comparative investigations showed that these values were 2- to 3-fold lower than those observed in the rat during the same period (Huntingdon Life Sci., Payan et al., 2003).

In vivo studies (animals)

After dermal exposure of NMP to rat skin at doses of 0.2, 2 and 20 mg/cm² applied to an area of 12 cm², there was 50 % absorption of the 2 lower doses while 75 % of the 20 mg/cm² dose was absorbed, suggesting that NMP promotes its own absorption. Maximum blood levels were observed approximately 8 hours after application (Research Triangle Institute, (Dermal administration), 1991). It was demonstrated that percutaneous absorption rate in rats was proportional to the concentration of NMP applied (Huntingdon, 1998) and was dependent on skin thickness. Maximum absorption fluxes of 9.7 mg/cm²/h (30 min) and 23.4 mg/cm²/h (45 min) NMP were determined for 20 µL/cm² and 40 µL/cm², respectively; absorption decreased when neat NMP was diluted (Payan et al., 2003).

Prolonged exposures to neat NMP (4 – 6 hr) can increase the permeability of the skin to NMP by as much as 7-fold (Payan et al., 2003). A study of the in vivo absorption of neat NMP under different occlusive conditions indicated that dermal absorption 1 hour post-exposure was greatest under unoccluded conditions (69 %), followed by semi-occluded (57 %) and occluded (50 %) conditions.The lower absorption seen with occlusion is considered to reflect dilution of the applied NMP by the transepithelial movement of water that subsequently becomes trapped at the site of occlusion (Inveresk Research, 2003).

In vivo studies

A mean 67.9 % absorption of NMP through the skin in 12 human volunteers exposed to 300 mg NMP via a skin patch was observed (Ligocka et al., Volunteer study, 2003). 12.6 % of the total dose was excreted as 5-HNMP, 6 - 12 hours after exposure, while 2-HMSI peaked at 2 time periods, 12 - 24 hours after exposure (3.3 % of dose) and 36 - 48 hours after exposure (3.2 % of dose).