Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, fully adequate for assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
- Identity: N-(2-Hydroxyethyl)-2-pyrrolidon
- Appearance: Clear, colourless to yellow liquid
- Batch No.: O 2909
- BASF Substance No.: 04/0443-2
- Purity: 99.7 area-% determinded by GC-analysis
- Homogeneity: The test substance appeared to be homogeneous
- Stability in Solvent: Not indicated by the sponsor
- Storage: At room temperature
- Expiration Date: August 21, 2012

In vivo test system

Test animals

other: CBA/CaCrl
Details on test animals and environmental conditions:
- Source: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom
- Age at study initiation: pre-test: 8-9 weeks, main study: 8-9 weeks
- Weight at study initiation:
- Housing: grouo housing in Makrolon Type II/III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) with granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water (e.g. ad libitum): Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: at least 5 days

- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

25, 50 and 100 %
No. of animals per dose:
Details on study design:
Three groups each of five female mice were treated with different concentrations (based on the results of a pretest) of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed using an analytical balance. Both ears were punched at the apical area and the punches were immediately weighed pooled per animal. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. The suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation) as well as for the ear weights, lymph node weights and lymph node cell counts.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Mean DPM per animal and S.I.
0 % (w/v): DPM = 460, S.I. = 1.00
5 % (w/v): DPM = 621.1, S.I. = 1.35
10 % (w/v): DPM = 1004.3, S.I. = 2.18
25 % (w/v): DPM = 3720.5. S.I. = 8.08

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see Remark
Concentration Stimulation index Result 0% 1.0 - 25% 1.14 negative 50% 0.22 negative 100% 1.47 negative
other: disintegrations per minute (DPM)
Remarks on result:
other: Concentration Disintegrations per minute 0 395.4 25% 451.8 50% 483.0 100% 579.4

Any other information on results incl. tables

- Viability / Mortality: No deaths occurred

- Clinical Signs: On day 3, al slight erythema of the ear skin was observed in the high dose group.

- Body Weights: No abnormalities recorded

- Ear Weights: No significant or biological relevant increase was observed.

Viability / Mortality

No deaths occurred during the study period.


Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.


Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.


Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weight or - cell count was not observed in any of the test item treated groups in comparison to the vehicle control group.


Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information