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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study OECD 429 with GLP compliance
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 10 weeks old (beginning of treatment)
- Weight at study initiation: 18.8 - 22.4 g
- Housing: group
- Diet: pelleted standard diet, ad libitum
- Water: 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: 22 +/- 2°C
- Humidity: 32 – 65% (acclimation period); 45 – 65% (main study)
- Air changes: about 10 / hour
- Artificial light: 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: 2012-01-11 To: 2012-02-08
Vehicle:
dimethylformamide
Concentration:
1) 25 %
2) 50 %
3) 100 %
No. of animals per dose:
5 (main Study)
Details on study design:
RANGE-FINDING TESTS:
A solubility experiment was performed in two female mice according to the recommendations given by OECD 429. The highest test item
concentration, which could be technically used was the undiluted test item (100%). Test item solution at a concentration of 50% was prepared
using dimethylformamide as vehicle.

At the tested concentrations (50 and 100 %) applied once daily each on three consecutive days to the dorsal ear surface, the animals did not show any signs of systemic toxicity. On day 3, both treated animals showed an
erythema of the ear skin (Score 1). On day 4-6, only the animal treated with the undiluted test item showed an erythema of the ear skin (Score 1).
Signs of excessive local skin irritation were not observed.

The test item in the main study was assayed at 25, 50 and 100% (w/w).

MAIN STUDY:
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay

CRITERIA USED TO CONSIDER A POSTIVIE REPONSE
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical
concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION
The test item was placed into an appropriate container on a tared balance and dimethylformamide was added to achieve the required test item
concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.
Concentrations were in terms of material as supplied. A concentration control analysis of all three doses was performed as a separate study
under the responsibility of the sponsor and the results were reported in a separate report.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25 %, 50 %
and 100 % (w/w) in dimethylformamide. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface of each ear once
daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the vehicle alone.

Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline (PBS) containing 20.2 μCi of 3HTdR (equivalent to
approximately 80.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. The draining
lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal) and weighted immediately using an analytical balance. Single cell
suspensions of pooled lymph node cells were prepared and after several washing steps the level of 3HTdR incorporation was measured by means of
liquid scintillation counting .

After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were
immediately weighed (pooled per animal) using an analytical balance.

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-sodium.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for the body weights, the ear weights, the lymph node weights and lymph node cell count as well as for the DPM values (group mean DPM ± standard deviation).

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 5% level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003). One outlier was identified. However, both biological and statistical significance were considered together.
Positive control results:
In the periodic positive control experiment with alpha-hexylcinnamaldehyde performed in December 2011 the EC3 (= estimated concentration for a
S.I. of 3) was calculated to be 14.4 %.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
S.I.: control (vehicle) : 1.00 25 % Delta-Valerolactone : 1.56 50 % Delta-Valerolactone : 1.23 100 % Delta-Valerolactone : 1.06 The EC3 value could not be calculated, because all S.I.s were below the threshold value of 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM per animal (mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group): control (vehicle) : 383.7 25 % Delta-Valerolactone : 600.1 50 % Delta-Valerolactone : 473.7 100 % Delta-Valerolactone : 405.1

Viability/Mortality

No death occured during the study.

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 nodes weights and cell count

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 weights or cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold.

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. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. None of the indices determined for the test item treated groups exceeded this threshold.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Local Lymph Node Assay

In a dermal sensitization study (Harlan CCR, 2012) with Delta-Valerolactone solved in dimethylformamide, female mice (CBA/CaOlaHsd) were tested using the method of Local Lymph Node Assay (LLNA). In this study, Stimulation Indices (S.I.) of 1.56, 1.23, and 1.06 were determined with the test item at concentrations of 25, 50, and 100% (w/w) indimethylformamid, respectively. An EC3 value was not calculated, because all S.I.s were below the threshold value of 3 for a positive response. A periodic positive control experiment was performed in December 2011 with alpha-Hexylcinnamaldehyde (vehicle: acetone: olive oil (4+1 v/v)), demonstrating the sensitivity and reliability of the experimental technique. Animals did not show any signs of systemic toxicity or local skin irritation during the study period. No deaths occurred during the study period.

Delta-Valerolactone was not a skin sensitiser under the test conditions of this study.


Migrated from Short description of key information:
The test item Delta-Valerolactone was found to be not a skin senitiser in the Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:
The key study was selected (GLP and guideline study).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification is not warranted according to the criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.