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Description of key information

A LLNA study was conducted according to OECD 429 using mouse (van Sas, 2017). Key study.

It was concluded that the test substance was considered to be a non-sensitizer under the conditions of the test.

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:
From 2017-02-15 to 2017-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Batch No.: APM0005851606003
Purity/Composition: 99.8% w/w
Test item storage: At room temperature
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 19.4 to 22.7 g
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 22°C
- Humidity: 42 to 45%.
- Air changes: At least 10 air changes per hour
- Photoperiod: 12-hours light and 12-hours dark
- IN-LIFE DATES: From: 2017-02-15 To: 2017-03-06
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-screen Test: 40% and 70% w/w
Main Study: 25%, 40% and 70% w/w
No. of animals per dose:
Pre-screen Test: two animals per dose
Main Study: five animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The highest concentration (70% w/w) was the highest concentration that could be prepared homogeneously.
- Irritation: no irritation
- Systemic toxicity: No signs of systemic toxicity were observed
- Ear thickness measurements: Variations in ear thickness during the observation period were less than 25% from Day 1 predose values.
- Erythema scores: 0

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: according to body weights
- Criteria used to consider a positive response: If the results indicate a SI > 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of Test Item: The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were stirred until and during dosing.
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 mL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 mCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the lab is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Value:
0.7
Test group / Remarks:
25% w/w dose group
Parameter:
SI
Value:
1.1
Test group / Remarks:
40% w/w dose group
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
70% w/w dose group
Cellular proliferation data / Observations:
- Skin Reactions / Irritation: No erythema was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 1 and 6, which did not hamper scoring of the skin reactions.
- Systemic Toxicity: One animal died during injection with thymidine due to a technical error. No further mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements: Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 40 and 70% were 334, 496 and 661 DPM, respectively. The mean DPM/animal value for the vehicle control group was 447 DPM.
- EC3 value: exceeds 70%.
Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicits a SI >= 3 when tested up to 70%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 70%.
Based on these results, test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice (Local Lymph Node Assay) after three epidermal exposures of the animals according to OECD 429.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 40 or 70% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No erythema was observed in any of the animals.

One animal died during injection with thymidine due to a technical error, no further mortality occurred.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 40 and 70% were 334, 496 and 661 DPM, respectively. The mean DPM/animal value for the vehicle control group was 447 DPM. The SI values calculated for the test item concentrations 25, 40 and 70% were 0.7, 1.1 and 1.5, respectively.

Since there was no indication that the test item elicits a SI  >= 3 when tested up to 70%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 70%.

Based on these results, test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation: Animal test give negative result (SI <3 (actual value 1.5)).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.4.2 (amending by Regulation (EC) No. 286/2011of 10 March 2011) the test substance is not classified as an sensitiser.