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Description of key information

Skin sensitisation

- Key study: According to OECD 429 and EU Method B.42. GLP study. There was no evidence suggesting that the test material acts as a sensitiser in mice. Therefore, it should not be regarded as a dermal sensitiser.

- Data waiving: an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Fron 29 March 2016 to 18 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to OECD and EU method with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Analytical purity: 91.5%
- Expiry date: 13 October 2015
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories GmbH, 5800 AN Venray The Netherlands. The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation:19-23 g
- Housing: Full barreir in an air-conditioned room
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test: lot nº.0631, main study lot nº. 0922
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, miccrobiological controls at regular intervals)
- Acclimation period: Adequate acclimatation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3ºC
- Humidity (%): 55+/-10%
- Air changes (per hr):at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12h light, 12h dark
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50% and 100% of test substance in the prescreen test.
25%, 50% and 100% of test substance in the main test.
No. of animals per dose:
2 animals / group in the prescreen test
5 animals / group in the main test. 3 test groups, 1 negative control group and 1 positive control group.
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test item was found to be 100%. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AAO.
- Irritation: An prescreen irritant test irritation was conducted.
- Systemic toxicity: Mice were observed daily for clinical signs of systemic toxicity or local irritation at the application site.
- Ear thickness measurements: Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximatelly 48h after the first measurement) and day 6
- Erythema scores: Both ears were observed for erythema score. Excessive local irritation was indicated by an erythema score >=3 and/or ear swellinf of <=25%

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT: The animals were randomly selected using the validated departamental computerised system E-WorkBook
- Name of test method: 3H-methyl thymidine incorporation method
- Criteria used to consider a positive response: A substance is regarded as sensitiser in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H--methyl-thymidine-incorporation into lymph node cells of the test animals, relative to that recorded for the lymph nodes of the control group animals (Stimulation Index equal to or greater than 3.0)
TREATMENT PREPARATION AND ADMINISTRATION: 3 test groups (3 different concentrations in AOO). Each mouse was treated by topical application of 25microL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three cosencutive days. The first treatment day is defined as study day 1 and the treatment duration was 5 days.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The stimulation index of the positive control was 5.7 and therefore the test is considered to be valid.
Key result
Parameter:
SI
Value:
1.6
Variability:
1.5-1.7
Test group / Remarks:
concentration of 25%
Key result
Parameter:
SI
Value:
1.7
Variability:
1.2-2.4
Test group / Remarks:
concentration of 50%
Key result
Parameter:
SI
Value:
1.3
Variability:
1.1-1.4
Test group / Remarks:
concentration of 100%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for the control group animals.

EC3 CALCULATION: EC3 values were derived by linear interpolation: EC3=c+[(3-d)/(b-d)]x(a-c) between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. EC3 value could not be calculated as the stimulation indices of all concentrations were below 3.

CLINICAL OBSERVATIONS: All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS: All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.
Interpretation of results:
GHS criteria not met
Conclusions:
There was no evidence suggesting that the test material acts as a sensitiser in mice. Therefore, it should not be regarded as a dermal sensitiser.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of the test material using an vivo LLNA test in mice. The procedure used is according to OECD Guideline 429, the EU Method B.42 and the EPA OPPTS 870.2600 with GLP. A prescreen test was performed in order to establish the solubility, general toxicity and local irritation potential of the test item. In the main test, five mice per group were treated with the test item at concentrations of 25% (v/v) or 50% (v/v) diluted in AOO or 100% (undiluted). Each mouse was treated daily by topical application of 25 µL to the entire dorsal surface of each ear during 5 days. The proliferative response was evaluated using the incorporated 3H-Methyl Thymidine determination method. The test item did not produce clinical signs, mortality or changes in body weight. The stimulation index was calculated for every treatment group and allowed to conclude that VL3 is expected to have no sensitizing potential properties and therefore should not be regarded as a dermal sensitizer.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
Study scientifically not necessary / other information available: an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available.
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A study was conducted to determine the skin sensitisation potential of the test material using an vivo LLNA test in mice. The procedure used is according to OECD Guideline 429, the EU Method B.42 and the EPA OPPTS 870.2600 with GLP. A prescreen test was performed in order to establish the solubility, general toxicity and local irritation potential of the test item. In the main test, five mice per group were treated with the test item at concentrations of 25% (v/v) or 50% (v/v) diluted in AOO or 100% (undiluted). Each mouse was treated daily by topical application of 25 µL to the entire dorsal surface of each ear during 5 days. The proliferative response was evaluated using the incorporated 3H-Methyl Thymidine determination method. The test item did not produce clinical signs, mortality or changes in body weight. The stimulation index was calculated for every treatment group and allowed to conclude that VL3 is expected to have no sensitizing potential properties and therefore should not be regarded as a dermal sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information, negative results in the OECD 429 test, the substance is not classified for sensitising properties in accordance with CLP Regulation (EU) No. 1272/2008.