2-pyrrolidone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study. The study was modified to a non radioactive procedure, but this has no impact on the result.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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)

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

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: not reported
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]
- Microbiological status of animals, when known: not reported
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: housed in fully air-conditioned rooms in which a central air-conditioning system
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: 15 days before the first test substance application
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h darkness
- IN-LIFE DATES: From: To: not reported

The single housed animals were identified by cage cards. The animals were housed in fully air-conditioned rooms in which a central air-conditioning system ensured a temperature in the range of 20 - 24 °C and a relative humidity in the range of 30 - 70 %. Deviations from these specifications that would have had an adverse effect on the test results did not occur. Illumination period was 12 h light and 12 h darkness. Acclimatization period 15 days before the first test substance application.

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:

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.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde, techn . 85%

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.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Test group / Remarks:

Test group which received 3 % preparations of the test substance

Remarks on result:

other: see Remark

Remarks:

In 3 %, 10 % and 50 % preparations of test substance Lymph Node Weight Index was 0.95, 0.92 and 1.20, respectively. Cell Count index: 0.99 (3 %), 10.1 (10 %) and 1.32 (50 %). Ear Weight Index is the same for all preparations: 1.05. All indices in control are 1.0.

Results 2

Parameter:

SI

Test group / Remarks:

Test group which received 10 % preparations of the test substance

Remarks on result:

other: see Remark

Remarks:

In 3 %, 10 % and 50 % preparations of test substance Lymph Node Weight Index was 0.95, 0.92 and 1.20, respectively. Cell Count index: 0.99 (3 %), 10.1 (10 %) and 1.32 (50 %). Ear Weight Index is the same for all preparations: 1.05. All indices in control are 1.0.

Results 3

Parameter:

SI

Test group / Remarks:

Test group which received 50 % preparations of the test substance

Remarks on result:

other: see Remark

Remarks:

In 3 %, 10 % and 50 % preparations of test substance Lymph Node Weight Index was 0.95, 0.92 and 1.20, respectively. Cell Count index: 0.99 (3 %), 10.1 (10 %) and 1.32 (50 %). Ear Weight Index is the same for all preparations: 1.05. All indices in control are 1.0.

Results 4

Parameter:

other: Lymph Node Weight Index

Value:

0.95

Test group / Remarks:

Test group which received 3 % prepartions of the test substance

Results 5

Parameter:

other: Lymph Node Weight Index

Value:

0.92

Test group / Remarks:

Test group which received 10 % prepartions of the test substance

Results 6

Parameter:

other: Lymph Node Weight Index

Value:

1.2

Test group / Remarks:

Test group which received 50 % prepartions of the test substance

Results 7

Parameter:

other: Cell Count Index

Value:

0.99

Test group / Remarks:

Test group which received 3 % preparations of the test substance

Results 8

Parameter:

other: Cell Count Index

Value:

1.01

Test group / Remarks:

Test group which received 10 % preparations of the test substance

Results 9

Parameter:

other: Cell Count Index

Value:

1.32

Test group / Remarks:

Test group which received 50 % preparations of the test substance

Results 10

Parameter:

other: Ear Weight Index

Value:

1.05

Test group / Remarks:

Test group which received 3 % preparations of the test substance

Remarks on result:

other: The statistically increased ear weights, which were concentration independent, indicate irritation of the ear skin at all concentrations.

Results 11

Parameter:

other: Ear Weight Index

Value:

1.05

Test group / Remarks:

Test group whcih received 10 % preparations of the test substance

Remarks on result:

other: The statistically increased ear weights, which were concentration independent, indicate irritation of the ear skin at all concentrations.

Results 12

Parameter:

other: Ear Weight Index

Value:

1.05

Test group / Remarks:

Test group whcih received 50 % preparations of the test substance

Remarks on result:

other: The statistically increased ear weights, which were concentration independent, indicate irritation of the ear skin at all concentrations.

Any other information on results incl. tables

Table 1: Stimulation index

Test group	Treatment	Lymph node Weight Index	Cell Count Index	Ear Weight Index
1	Vehicle	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

The statistical evaluations were preformed using the WILCOXON-test( # for p<= 0.05 ## for p<= 0.01).

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. 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

1-ethylpyrrolidin-2-one (NEP) is negative in the Local Lymph Node Assay.

Executive summary:

To determine the sensitizing potential of 1-ethylpyrrolidin-2-one (NEP) was applied in three different concentrations (3 %, 10 % and 50 % in acetone) to the dorsum of both ears of mice for three consecutive days. On day six the lymph nodes were removed, and number of proliferated lymphocytes were counted. Thereafter the stimulation indices were calculated. The cell count and the lymph node index were slightly increased only in the highest preparation group. This observation is considered not to be treatment related. The test material is not sensitizer in this assay.