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
Study period:
19/01/2014 - 18/03/2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to guideline under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the UK, 30 November 2012
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Substance type: UVCB
- Physical state: paste
- Purity: 100% (UVCB)
- Storage condition of test material: room temperature in dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/CaOIaHsd
- Source: Harlan Laboratories Uk Ltd., Oxon, UK
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were grouped housed in suspended solid floor polypropylene cages furnished with softwood wood flakes
- Diet: Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Acclimatization Period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (degrees C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hour): Approximately 15
- Photoperiod: 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary Test: 100%
Main Test: 25% and 50% v/v in acetone/olive oil ( 4:1) and 100%
No. of animals per dose:
5
Details on study design:
Preliminary test :
The mouse was treated by daily application of 25 μL of the undiluted test item, to the dorsal surface of each ear for three consecutive days
(Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test :
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methylthymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Observations :
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal procedures :
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the
pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4 C, the precipitates were recovered by centrifugation at 2100 rpm(approximately 450 g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by -scintillationcounting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken
vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
α-Hexylcinnamaldehyde, tech., 85% showed a Stimulation Index (SI) of 8.42 in positive control study at a 25% concentration in acetone/olive oil 4:1.
α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Test item (25% in acetone/olive oil 4:1): 1.26 Test item (50% in acetone/olive oil 4:1): 2.37 Test item (100%): 2.62
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 18518.67 Test item (25% in acetone/olive oil 4:1): 23272.92 Test item (50% in acetone/olive oil 4:1): 43905.08 Test item (100%): 48496.46

Any other information on results incl. tables

Preliminary test:

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on these results the concentrations used in the main test were chosen.

Main test:

- Clinical observations: No deaths were observed. No signs of systemic toxicity in all were noted during the test.

- Body weight: Body weight change of the test animals between Day 1 and 6 was comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance is considered to be not sensitising
Executive summary:

Exposure of 4 mice/ treatment during 3 consecutive days at the dorsal site of the ears with test substance (25%, 50% [in acetone/olive oil 4:1] or 100%) or vehicle. On day 6, 3H-methyl thymidine(20 μCi/mouse) was injected in the tail vein. Five hours thereafter mice were killed and draining auricular lymph nodes were pooled per dose group and assessed for 3HTdR after 18 hours incubation at 4°C using a Beckman LS6500 scintillation system.

At the highest dose tested the stimulation index was 2.62. The test substance is considered to be not sensitizing to the skin.