Registration Dossier

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 guidelien study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Pentaerythritol, olig. react. prod. with 1-chloro-2,3-epoxypropane, react. prod. with acrylic acid
- Physical state: liquid, viscous/yellowish
- Analytical purity: 100 %
- Lot/batch No.: Mischcharge aus G44/082/11 und G44/085/11

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, Borchen, Germany
- Age at study initiation: 8 weeks
- Housing: single housing (Makrolon gabe, type II)
- Diet (e.g. ad libitum): STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 -70 %
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 10 and 25%
No. of animals per dose:
5
Details on study design:
Three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in N, N-dimethylformamide by topical application to the dorsum of each ear for three consecutive days.
The application volume 25 µL/ear/day was spread over the entire dorsal surface (Ø 8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25 % α-hexyl cinnamaldehyde dissolved in N, N-dimethylformamide.
Four days after the first topical application, the mice were intraperitoneally injected with BrdU. Approximately 24 hours after intraperitoneally injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed using an analytical balance. Furthermore, both ears of mice were punched at the apical area using a biopsy punch and the punches were immediately weighed pooled per animal using an analytical balance. 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 proliferative capacity of pooled lymph node cells was determined by the incorporation of BrdU measured in a photometer.

Results and discussion

Any other information on results incl. tables

A statistically significant increase in ear weights was observed in all doses groups, but was considered not to be biologically relevant. According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

In this study Stimulation Indices (S.I.) of 1.0, 1.1 and 1.9 were determined with the test item at concentrations of 5, 10 and 25 % (w/w) in N, N-dimethylformamide, respectively. Based on the S.I.s obtained with 10 and 25% test item concentration, an EC1.6 value of 19.38% (w/w) was calculated. A clear dose response was observed. A statistically significant and biologically relevant increase in BrdU labeling was observed for the high (25%) dose group. Also observed was a statistically significant increase in lymph node weights for the low (5%) and high (25%) dose group and in lymph node cell count for the mid (10%) and high (25%) dose group. Furthermore, the cut-off-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice (see Ref. 8) was exceeded in the high dose group.

Applicant's summary and conclusion