Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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 guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Geranylacetate Extra
- Physical state: colourless, clear liquid
- Analytical purity: 98.8 corr. area-% (GC-analysis)
- Batch No.: Ch 00006277L0
- Expiration date of the batch: 04 February 2015
- Storage condition of test material: refrigerated, under light exclusion, air sensitive

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories B.V.,The Netherlands
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 20.0 to 24.9 g (weight variation does not exceed ± 20% of the mean weight at beginning of treatment)

- Housing: group housed in Makroion cages with stainless steel mesh lids and fumished with granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45 - 65
- Air changes (per hr): min. 8
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50, 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TEST:
Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% (undiluted) once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance.
At the tested concentrations the animals did not show any signs of excessive local skin irritation or systemic toxicity. Thus, the test item in the main study was assayed at 25, 50, and 100%.

TREATMENT PREPARATION AND ADMINISTRATION IN THE MAIN EXPERIMENT:
Each test group of mice were treated by topical application to the dorsal surface of each ear. The application volume, 25 µl/ear/day, was
spread over the entire dorsal surface (diameter: 8 mm) of each ear once daily for three consecutive days. A further group of mice (vehicle control animals) was treated with an equivalent volume of the relevant vehicle alone.

Five days following the first topical application of the test item (day 6) 250 µL of phosphate buffered saline (PBS) containing 79.8 µCi/mL 3H-methyl thymidine (3HTdR) were injected into each mouse via the the tail vein.

Approximately five hours after the administration of 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal) and weighed immediately using an analytical balance. Single cell suspensions (in PBS) of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 200 µm-mesh stainless steel gauze. The level of 3HTdR incorporation was measured in a ß-scintillation counter. Furthermore, the lymph node cell count was determined for the suspensions of each animal using a cell counter.

All animals were observed at least once daily from experimental start to necropsy. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study was recorded. Body weights were recorded on day 1 (prior to dosing) and prior to treatment with 3HTdR. After the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal using an analytical balance.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle control: 1.0 25% test substance: 4.95 50% test substance: 7.33 100% test substance: 7.56
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal (SD) Vehicle control: 380.9 (126.3) 25% test substance: 1885.1 (440.2) 50% test substance: 2791.1 (1461.4) 100% test substance: 2879.3 (421.4)

Any other information on results incl. tables

No deaths occurred during the study period.

No signs of systemic toxicity or local skin irritation were observed during the study period.

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.

Ear weights:
Test item concentration %(w/w) Mean Ear weight (mg) SD Index
(Value Test Group versus
Value Control)
Vehicle Control (AOO) 25.1 1.5 1.0
25% 27.1 * 1.0 1.08
50% 27.3 * 1.0 1.09
100% 26.9 * 0.7 1.07

Lymph node cell counts:

Test item concentration %(w/w) Mean Lymph Node Cell Count x10E06 per animal SD Index
(Value Test Group versus
Value Control)
Vehicle Control (AOO) 9.9 1.9 1.0
25% 21.9 * 2.3 2.2
50% 31.1 * 9.5 3.1
100% 25.1 * 2.7 2.5

Lymph node weights:

Test item concentration %(w/w) Mean lymph node weight (mg) SD Index
(Value Test Group versus
Value Control)
Vehicle Control (AOO) 5.3 0.6 1.0
25% 9.0 * 0.6 1.7
50% 10.6 * 1.9 2.0
100% 10.0 * 0.7 1.9

*=significant

A statistically significant increase in ear weights was observed in all dose groups in comparison to the vehicle control group (p≤0.05). Furthermore, the cut-off value of 1.1 of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was almost reached in all dose groups (indices of 1.08, 1.09, and 1.07, respectively), indicating a borderline positive response for ear skin irritation.

In this study Stimulation Indices (S.I.) of 4.95, 7.33 and 7.56 were determined with the test item at concentrations of 25 and 50 (w/w) in acetone:olive oil (4+1 v/v), and 100%, respectively. A clear dose response was observed. A statistically significant and biologically relevant increase in DPM values was observed in all test groups in comparison to the vehicle control group and all S.I. determined exceeded the threshold value of 3 for a positive response.

Furthermore, a statistically significant and biologically relevant increase in lymph node cell count and also lymph node weights was observed in all dose groups in comparison to the vehicle control group (p≤0.05). For BALB/c mice, a cut-off –value for the lymph node cell count index of 1.55 was reported for a positive response. According to this criterion, the lymph node cell count indices determined for all dose groups exceeded this threshold value (indices of 2.2, 3.1, and 2.5, respectively).

Applicant's summary and conclusion