Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with OECD GLP (1997) regulations and OECD Guideline 429 (2010).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Viscous liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 15655
- Substance type: Mono-constituent
- Physical state: Viscous liquid
- Analytical purity: 91.4%
- Purity test date: 21 April 2015
- Lot/batch No.: 565093
- Expiration date of the lot/batch: 27 August 2017

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: 19-24 grams
- Housing: Group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilized sawdust as bedding.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 April 2015 To: 04 May 2015

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50, or 100% test substance
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Soluble in vehicle
- Irritation: No irritation was observed in any animals in the pre-screen test up to 100% test article.
- Lymph node proliferation response: Not examined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: A Stimulation Index (SI) greater than 3 indicates a positive response.

TREATMENT PREPARATION AND ADMINISTRATION: The test substance preparations were prepared within 4 hours of dosing. The dorsal surface of both ears was topically treated (25 uL/ear) with the test substance at approximately the same time on each day for three consecutive days (Study Days 1, 2, and 3). 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 substance. On Study Day 6, each animal was injected via tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 uCi 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 approximately 3 mL PBS. Following node excision, a single cell suspension of local lymph node cells was prepared in PBS by gentle separation through stainless steel gauze. Lymph node cells were washed twice in PBS by centrifugation at 200g for 10 minutes at 4 C. To precipitate the DNA, the lymph node cells were exposed to 5% tricholoroacetic acid and then stored in the refrigerator until the next day. On Study 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. Counts were converted to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
6 month reliability checks show consistent positive results.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the test substance concentrations of 25, 50, and 100% were 1.3, 1.7 and 1.2, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with the test substance concentrations 25, 50, and 100% were 590, 802, and 543 DPM, respectively. The mean DPM/animal value for the vehicle control group was 461 DPM.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of the study, the test article showed no evidence of dermal sensitization potential (SI=1.2 at 100% test article).
Executive summary:

The dermal sensitization potential of the test article (Viscous yellow liquid, Lot: 565093, Purity 91.4%) was evaluated in the local lymph node assay (LLNA) with CBA/J strain mice. The study was performed in compliance with OECD GLP ENV/MC/CHEM (98) 17 (revised 1997). The test method was based on OECD Section 4 No. 429 (2010), EC No. 440/2008 B42, and EPA OPPTS 870.2600 (2003). The test article was diluted in acetone/olive oil (4:1 v/v) as needed. A pretest was conducted and the highest test article concentration (100%) was selected as the high dose for the main phase. Female mice (5/treatment) received the control (acetone/olive oil), 25%, 50%, or 100%, concentrations of the test substance. The corresponding treatment (25 uL/ear) was applied to the dorsal surface of both ears for three consecutive days. Three days after the last exposure, all animals were injected with 0.25 mL sterile phosphate buffered solution containing 3 H-methyl thymidine and subsequently euthanized. The auricular lymph nodes were removed to visually estimate the relative size and to observe any abnormalities. The nodes were pooled for each animal to measure the amount of operative DNA by disintegrations per minute (DPM). The stimulation index (SI) was calculated for each group. The 6-month reliability check with alpha-hexylcinnamicaldehyde indicated that the LLNA is an appropriate model for evaluating dermal sensitization at the test facility. Observations for mortality (twice daily), body weights (Day 1 and Day 6), clinical signs (once daily), and irritation (once daily) were performed as well. Mean DPM/animal values were 590, 802, and 543 DPM for the 25%, 50%, and 100% test article concentrations, respectively. The control mean DPM/animal value was 461 DPM. The SI values were 1.3, 1.7, and 1.4 for the 25%, 50%, and 100% test concentrations, respectively. No irritation of the ears was observed in any animal. No mortality, clinical signs of toxicity, or significant body weight changes were observed. All auricular lymph nodes of the test and control animals were considered normal in size. Based on the results of the study, the test article showed no evidence of a dermal sensitization potential (SI=1.2 at 100% test article).