1-methyl-2-pyrrolidone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-04-26 to 2005-07-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

other: Amendment

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

(April 24, 2002)

Deviations:

yes

Remarks:

non radioactive method, but no impact on the result

Principles of method if other than guideline:

The Murine Local Lymph Node Assay (LLNA) is recommended by international test guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanatalt for Pflanzenbau und Pflanzenschutz, Essenheimer Str. 144, D-55128 Mainz, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-ethylpyrrolidin-2-one

EC Number:

220-250-6

EC Name:

1-ethylpyrrolidin-2-one

Cas Number:

2687-91-4

Molecular formula:

C6H11NO

IUPAC Name:

1-ethylpyrrolidin-2-one

Details on test material:

- purity: 99.8 corr. area-%
- Batch No.: 29151288P0

Specific details on test material used for the study:

- purity: 99.8 corr. area-%
- Batch No.: 29151288P0

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 16.4 g - 21.0 g
- Housing: single
- Diet: ad libitum, Kliba-Labordiät ( Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland)
- Water: ad libitum, tap water
- Acclimation period: 15 days

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

Study design: in vivo (LLNA)

Vehicle:

other: acetone

Concentration:

50 %, 10 % and 3 % w/w preparations of the test substance in acetone

No. of animals per dose:

6

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
The study comprised three treatment groups and a vehicle control group. Each group consisted of 6 mice.

TREATMENT PREPARATION AND ADMINISTRATION
APPLICATION
Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

EAR WEIGHT
Immediately after the death of each animal a circular piece of tissue (diameter 0 .8 cm) was punched out of the apical part of each ear.

LYMPH NODE WEIGHT
Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.

PREPARATION OF CELL SUSPENSION AND DETERMINATION OF CELL COUNT
After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 pm) into 6 mL of phosphate-buffered physiological saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1 :500. The cell count was determined using a Casy-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of lymph node weight, cell count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group. The Wilcoxon test was used to examine the lymph node weight, cell count and ear weight.

Results and discussion

Positive control results:

A study with 1%, 3% and 10% of Alpha-Hexylcinnamaldehyde (techn. 85%) in acetone was performed between 2005-04-26 and 2005-05-10 to show that the test system is able to detect sensitizing compounds.
The results and discussion was:

No signs of systemic toxicity were noticed . The test substance induced a statistically significant and biologically relevant response in the auricular lymph nodes, when applied as 3% or 10% preparations in acetone. The 1% test substance preparation caused a statistically significant increase in cell counts, which, due to its minor extent, is not considered to be biologically relevant . The limited but statistically significant increases in ear weights indicate some irritation of the ear skin at 3% and 10% .
This mild irritation, however, does not explain the distinct lymph node responses observed with these concentrations.

Thus it is concluded that the Murine Local Lymph Node Assay is able to show the skin sensitizing effect of Alpha-Hexylcinnamaldehyde, techn. 85% under the test conditions chosen.
A concentration of 1% is considered to represent a threshold for sensitization induction in this test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

other: cell count index

Value:

0.99

Test group / Remarks:

3% in acetone

Results 2

Key result

Parameter:

other: cell count index

Value:

1.01

Test group / Remarks:

10% in acetone

Results 3

Key result

Parameter:

other: cell count index

Value:

1.2

Test group / Remarks:

50% in acetone

Remarks on result:

other: statistically significant but not biologically relevant, irritation of ear skin occurred

Results 4

Parameter:

other: Lymph node weight index

Value:

>= 0.92 - <= 0.95

Test group / Remarks:

3% and 10% preparation in acetone / not statistically significant or biologically relevant

Results 5

Parameter:

other: Lymph node weight index

Value:

1.2

Test group / Remarks:

50% preparation in acetone / statistically significant but not biologically relevant

Results 6

Parameter:

other: Ear weight index

Value:

1.05

Variability:

0.4 - 0.7

Test group / Remarks:

3%, 10% and 50% preparation in acetone / dose indipendet, biologically indication of irritation

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
- no induction of a statistically significant or biologically relevant response of the auricular lymph nodes for 3% or 10% preparations
- minimal but statistically significant increase in mice treated with the 50% test substance preparation not considered biologically relevant

EAR WEIGHTS:
- 3%, 10% or 50% preparations in acetone induced statistically significant increases in ear weight
- increases were concentration independent

CLINICAL OBSERVATIONS:
- no abnormalities observed

BODY WEIGHTS:
- expected body weight gain was generally observed in the course of the study

Any other information on results incl. tables

Table 1: Results of the main LLNA study with NMP solutions.

Test group	Treatment	Lymph node weight Index	Cell count Index	Ear Weight Index
1	Vehicle alone	1.00	1.00	1.00
2	3 % in acetone	0.95	0.99	1.05#
3	10 % in acetone	0.92	1.01	1.05#
4	50 % in acetone	1.20#	1.32#	1.05#

# for p <= 0.05; ## for p <= 0.01

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of systemic toxicity were noticed .

The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as 3% or 10% preparations in acetone. The minimal but statistically significant increase in mice treated with the 50% test substance preparation was too small to be considered biologically relevant. The statistically significant increases in ear weights, which were concentration independent, indicate irritation of the ear skin at all concentrations.

Thus, the minimal increase in cell count and lymph node index in test group of the highest dose is considered is to be related to ear skin irritation produced by the combination of vehicle and test substance, which starts already at the 3% concentration without relevant lymph node response.
Higher concentrations were not tested, because the high concentration tested (50%) produced ear skin irritation.

Based on the results discussed above and applying the evaluation criteria cited in above (refer to 'Any other information on material and methods incl. tables'), it is concluded that N-Ethyl-2-pyrrolidon does not have a sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of N-Ethyl-2-pyrrolidon was assessed using the nonradioactive variant of the Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears.

Groups of 6 female CBA/Ca mice each were treated with 50 %, 10 % and 3 % w/w preparations of the test substance in acetone or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as 3 % or 10 % preparations in acetone. The minimal but statistically significant increase in mice treated with the 50 % test substance preparation was too small to be considered biologically relevant. The statistically significant increases in ear weights, which were concentration independent, indicate irritation of the ear skin at all concentrations. Thus, the minimal increase in cell count and lymph node index in test group 4 is considered to be related to ear skin irritation produced by the combination of vehicle and test substance, which starts already at the 3% concentration without relevant lymph node response.

From the results of the study it is concluded, that N-ethyl-2-pyrrolidone does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.