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Basic toxicokinetics

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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication which meets basic scientific principles
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication which meets basic scientific principles
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
In-vivo percutaneous absorption test
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River breeding Laboratories (Portage, MI, USA)
- Age at study initiation: not stated
- Weight at study initiation: 192-239 g
- Fasting period before study: not specified
- Housing: individual
- Individual metabolism cages: yes
- Diet: LAD1 Bioscure Ltd., Manea, UK ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
other: isopropanol
Duration of exposure:
single exposure, up to 120 hrs
Doses:
- Nominal doses: Males: ca. 15 mg/ animal (1.67 mg/cm²) Females: ca. 15 mg/ animal (1.67 mg/cm²)
No. of animals per group:
33 males and 33 females in total; 3 per sex and time-point
Control animals:
yes
Remarks:
vehicle
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: each compound was diluted with the corresponding non-radioactive compound and, where necessary, purified by distillation to provide material of above mentioned purity.
- Method of storage: not reported

APPLICATION OF DOSE: to an area of 9 cm² of carefully-shaved skin on the back of each animal

VEHICLE
- Justification for use and choice of vehicle (if other than water): isopropanol
- Amount(s) applied (volume or weight with unit): 150 µL
- Concentration (if solution): mixture 22.5 mg/15 mg (see above) in 150 µL isopropanol
- Lot/batch no. (if required): not reported
- Purity: not reported

TEST SITE
- Preparation of test site: shaved
- Area of exposure: back
- % coverage: not reported
- Type of cover / wrap if used: aluminium foil
- Time intervals for shavings or clipplings: not reported

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: adhesive tape was used to secure the aluminium foil

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: after the animals were killed
- Washing procedures and type of cleansing agent: the occlusive dressing from each topically-dosed rat was removed and incubated (40 °C, 24h) with 100 mL of the digestion mixture (methanol/aqueous methanol (50%))
- Time after start of exposure: 120 h

SAMPLE COLLECTION
- Collection of blood: by cardiac puncture
- Collection of urine and faeces: at the final killing time. The metabolism cages were washed with water, and the washungs, excreta and carcasses stored
- Collection of expired air: at the final killing time, expired air was trapped in a mixture of ethanolamine-2-ethoxyethanol (1:3, v/v) at 24h intervals
- Terminal procedure: the rats were killed by cervical dislocation
- Analysis of organs: yes. Remaining carcasses from topically-dosed rats were solubilized by digestion (45 °C, 48 hr) in a solution of NaOH (80g) and Triton X-405 (p-tert-octylphenoxypolyethoxyethanol; 100 mL) in methanol (300 mL) diluted to 1 litre with distilled water.

SAMPLE PREPARATION
- Storage procedure: at -20 °C until analysed
- Preparation details: Replicate aliquots (0.25-1 mL) of plasma, urine, cage washings, contents of expired air traps, occlusive dressing digests and extracts, application-site washings, skin digests and carcass digests were separately mixed with Special Scintillator MI-31 (Canberra Packard, Pangbourne, UK) for measurement of radioactivity. Replicate weighed portions of faeces (after homogenization with distilled water) were combusted in oxygen using an automatic sample oxidizer (Packard Model 306), and the products of combustion dissolved in Optisorb 1 and mixed with Optisorb S scintillator (FSA Laboratory Supplies, Loughborough, UK) for measurement of radioactivity.

ANALYSIS
- Method type(s) for identification (HPLC-UV for measurement of unchanged test substances in the plasma, Liquid scintillation counting for measurement of radioactivity)
- Liquid scintillation counting results (cpm) converted to dpm as follows: not reported
- Validation of analytical procedure: yes
- Limits of detection and quantification: not reported
Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
- Non-occlusive cover + enclosure rinse: done, but results were not reported
- Skin wash: done, but results were not reported
- Skin test site: done, but results were not reported
- Skin, untreated site: done, but results were not reported
- Blood: Following dermal co-administration, plasma radioactivity levels changed little during the first six hours. The greatest mean values were obtained at 2h (0.265 % dose/mL) and 6 h (0.204 % dose/mL) after dosing. 12 hours following topical administration little or none of the intact pyrrolidinones were found in plasma
- Carcass: 1 % of the total dose remained in the carcass
- Urine: Urinary excretion was predominant (61-70 %) mostly during the first 24 h after dosing.
- Cage wash + cage wipe: done, but results were not reported
- Faeces: Faecal excretion represented only 1-2 % of the total dose
- Expired air (if applicable): 6-7 % was excreted by exhalation
Total recovery:
up to 93 %
Dose:
6.15 µg/mL
Parameter:
percentage
Absorption:
ca. 0.1 %
Remarks on result:
other: 6 h
Remarks:
Time point is the time taken to attain dose (males)
Dose:
10.93 µg/mL
Parameter:
percentage
Absorption:
0.1 %
Remarks on result:
other: 2 h
Remarks:
Time point is the time taken to attain dose (female)
Conversion factor human vs. animal skin:
not appplicable

Results on pharmakokinetic parameters are attached in "Attached background material".

Table 2: Excretion balance of radioactivity following co-administration of 14C-NMP and 14C-2 -P to rats

Oral

Dermal

Sample

Time (hr)

M

F

M

F

Urine

0-6

16.0

21.4

5.5

11.0

6-24

65.2

60.9

45.3

46.7

24-72

2.9

4.2

8.1

8.8

72-120*

1.1

1.3

2.4

3.3

Total

85.2

87.8

61.3

69.8

Faeces

0-24

1.2

0.6

0.7

0.5

24-72

0.8

0.5

0.8

0.6

72-120

0.1

0.1

0.1

0.2

Total

2.1

1.2

1.6

1.3

Expired air

0-24

6.8

4.8

3.5

4.2

24-72

0.6

0.6

2.0

2.0

72-120

0.1

0.1

0.5

0.5

Total

7.5

5.5

6.0

6.7

Total excretion

0-120

94.8

94.5

68.9

77.8

Carcass

120

1.1

1.2

1.0

1.1

Dose dressing

120

10.7

11.3

Dose-site wash

120

1.2

0.3

Treated skin

120

11.2

2.0

Untreated skin

120

ND

<0.1

Total recovery

0-120

95.9

95.7

93.0

92.5

Conclusions:
The compoment 2-pyrrolidone of the mixture of N-methyl-2-pyrrolidinone (NMP) and 2-pyrrolidinone (2-P) administered topically is absorbed through the skin and then mainly excreted through the urine. Percutaneous absorption is greater in femals than in males (Table 2).
Executive summary:

Male and female Sprague-Dawley CD strain (albino) rats were administered topically to the [14C]NMP/[14C]2-P mixture (22.5mg/15mg) to assess percutaneous absorption and excretion rates of these compounds.

The radioactivity was measured in plasma, urine, faeces, expired air and remaining carcasses of killed animals to caculate pharmacokinetic parameters of the applied mixture and excretion balance.

Mean concentrations of 2 -pyrrolidone in plasma reached values after 6 h for males and 2 h for females However, plasma concentration of the unchanged 2 -pyrrolidone altered little during 6 -h post-administration and reflected that of total plasma radioactivity. At the termination, plasma level of the unchanged 2 -pyrrolidone was not measurable anymore.

The balance of the radioactivity applied was recovered in the all samples taken. The excretion of 2 -pyrrolidone take place predominantly through the urine (Table 2 in "Remarks on results").

Data source

Reference
Reference Type:
publication
Title:
Percutaneous absorption of co-administered N-methyl-2-[' 4C]pyrrolidone and 2-['4C]pyrrolidone in the rat
Author:
Midgley I, et al.
Year:
1991
Bibliographic source:
Fd Chem. Toxic., Vol. 30(1), 57-64

Materials and methods

Objective of study:
absorption
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The oral absorption of a mixture of N-methyl-2-pyrrolidinone and 2-pyrrolidinone has been investigated in rats in vivo.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-pyrrolidone
EC Number:
210-483-1
EC Name:
2-pyrrolidone
Cas Number:
616-45-5
Molecular formula:
C4H7NO
IUPAC Name:
pyrrolidin-2-one
Constituent 2
Chemical structure
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
Details on test material:
- Name of test material (as cited in study report): 2-pyrrolidinone and N-methyl-2-pyrrolidinone, mixture (2:3)
- Physical state: liquid
- Specific activity (if radiolabelling): 4.9 (2-P) and 7.4 mCi/mmol (NMP)
- Locations of the label (if radiolabelling): N-methyl-2-[2-14C]pyrrolidinone; 2-[5-14C]pyrrolidinone
Radiolabelling:
yes
Remarks:
2-[14C]pyrrolidone

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River breeding Laboratories (Portage, MI, USA)
- Age at study initiation:
- Weight at study initiation: 192-239 g
- Fasting period before study: not reported
- Housing: individual
- Individual metabolism cages: yes
- Diet: LAD1 Bioscure Ltd., Manea, UK ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: water for oral adminstration and isopropanol for dermal administration
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
[14C]NMP / [14C]2-P mixure (22.5 mg / 15 mg) was prepared as an aqueous mixture (0.6 mL);

These doses were equivalent to about 112 mg NMP and 75 mg 2-P /kg bw
Duration and frequency of treatment / exposure:
single exposure, up to 120 hrs
Doses / concentrations
Remarks:
Doses / Concentrations:
ca. 112 mg NMP and 75 mg 2-P /kg bw
No. of animals per sex per dose / concentration:
33 males and 33 females in total; 3 per sex and time-point
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
not done
Details on study design:
- Dose selection rationale: no data
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum, cage washes, bile, CO2
- Time and frequency of sampling: 24h intervals (expired air); other parameters at termination of animals

Sample collection:
Following blood withdrawal by cardiac puncture under halothane anaesthesia, the rats were killed by cervical dislocation in groups of six (three males, three females) per time point at selected times after dosing. Plasma was separated from the blood cells by centrifugation, and stored at about -20 °C until analysed. Urine and faeces were collected at suitable intervals from the six rats in each group killed at the final killing time (i.e. 120 hr), and any [14C]CO2 present in the expired air was trapped in a mixture of ethanolamine-2-ethoxyethanol (1:3, v/v) at 24-hr intervals. After these rats were killed, the metabolism cages were washed thoroughly with water, and the washings, excreta and carcasses stored at about -20 °C until analysed.
Statistics:
data not available

Results and discussion

Preliminary studies:
not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Absorption and distribution:
After oral administration, the concentrations of radioactivity were greatest in plasma of male and female rats at 2 hrs after dosing (mean values 0.463 % and 0.562 % dose/mL). Radioactivity declined thereafter in a muliphasic manner with a terminal half-life of 29 and 27 hrs for male and female rats. The concentrations were still measureable 5 days after dosing when the mean values corresponded to 0.0006 % of the total dose in both sexes.

Quantitative analysis of plasma by HPLC showed, that at 30 min after dosing the proportions of unchanged NMP and 2-P reflected the 3: 2 ratio, demonstrating little metabolism at this time point. Both parent compounds together accounted for at least 80% of plasma radioactivity until 8 hrs post-administration indicating little first-pass metabolism.
Details on distribution in tissues:
not measured
Details on excretion:
Metabolism and excretion:
Biotransformation became more evident 8 hours post-administration and by 12 hrs virtually all of the plasma radioactivity was found in form of unknown polar metabolites. Plasma levels of the unchanged pyrrolidinones were not measurable during the terminal phase of plasma radioactivity disposition after oral dosing.
Following oral co-administration to male and female rats, 94.8 % and 94.5 % of the radioactive dose were excreted within 5 days. Radioactivity was mainly excreted through the urine (85-88 % of the total dose) mostly within 24 hours. Faecal excretion was of minor importance representing only 1-2 % of the total dose. 6-7 % of the oral dose was eliminated as CO2 in the expired air, which indicates the extent of metabolic degradation of the pyrrolidinone ring. Only about 1 % of the total dose remained in the carcass, demonstrating that the excretion was virtually complete by the end of 5 days post-exposure. Recovery of radioactivity in excreta is presented in Table 2 in "Remarks on results".
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
Tmax: 2h after oral route of administration
Test no.:
#2
Toxicokinetic parameters:
Tmax: 6 and 2 h for males and females, respectively after dermal administration
Test no.:
#1
Toxicokinetic parameters:
Cmax: 0.171 and 0.215 % dose/mL for m/f respectively after oral administration
Test no.:
#2
Toxicokinetic parameters:
Cmax: 0.052 and 0.093 % for m/f respectively after dermal administration
Test no.:
#1
Toxicokinetic parameters:
AUC: 1.07 and 1.33 % dose/mL h after oral administration
Test no.:
#2
Toxicokinetic parameters:
AUC: 0.31 and 0.59 % dose/mL h after dermal administration

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
12 hours following oral administration little or none of the intact pyrrolidinones were found in plasma and virtually all of the radioactivity was associated with unknown polar metabolites.

Any other information on results incl. tables

The only results of measured radioactivity of 2 -pyrrolidone in plasma are presented in "Toxicokinetic parameters". All pharmakokinetic parameters are presented in Table 1 atttached in "Attached background material".

Table 2: Excretion balance of radioactivity following co-administration of 14C -NMP and 2 -P to rats

Oral

 

Dermal

Sample

Time (hr)

M

F

M

F

Urine

0-6

16.0

21.4

5.5

11.0

6-24

65.2

60.9

45.3

46.7

24-72

2.9

4.2

8.1

8.8

72-120*

1.1

1.3

2.4

3.3

 

Total

85.2

87.8

61.3

69.8

Faeces

0-24

1.2

0.6

0.7

0.5

24-72

0.8

0.5

0.8

0.6

72-120

0.1

0.1

0.1

0.2

 

Total

2.1

1.2

1.6

1.3

Expired air

0-24

6.8

4.8

3.5

4.2

24-72

0.6

0.6

2.0

2.0

72-120

0.1

0.1

0.5

0.5

 

Total

7.5

5.5

6.0

6.7

Total excretion

0-120

94.8

94.5

68.9

77.8

Carcass

120

1.1

1.2

1.0

1.1

Dose dressing

120

10.7

11.3

Dose-site wash

120

1.2

0.3

Treated skin

120

11.2

2.0

Untreated skin

120

ND

<0.1

Total recovery

0-120

95.9

95.7

93.0

92.5

Applicant's summary and conclusion

Conclusions:
The compoment 2-pyrrolidone of the mixture of N-methyl-2-pyrrolidinone (NMP) and 2-pyrrolidinone (2-P) is considered to be fully excreted within 5 days.
Executive summary:

The percutaneous absorption has been investigated in rats of a mixture (3:2, w/w) of N-methyl-2-pyrrolidinone (NMP) and 2-pyrrolidinone (2-P). Co-administration of the two 14 C-radiolabelled compounds was performed by the dermal and oral routes.

Radioactivity was excreted predominantly through the urine after either route of administration, and comparison of the respective excretion profiles indicated that about three-quarters of the applied dose was absorbed through the skin. NMP appeared to be absorbed through the skin more extensively and at a slightly faster rate than 2-P; total percutaneous absorption tended to be more extensive in female than in male rats.

Only about 1 % of the dose was retained in the carcasses, demonstrating that the excretion of radioactivity was virtually complete by the end of the 5-day collection period.