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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the O.E.C.D. test guideline 429 with GLP compliance.

Data source

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

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Colorless liquid at room temperature.
Details on test material:
As per IUCLID5 Sections 1.1. 1.2. and 4.1.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. After an acclimatization period of at least five days the animals were selected and placed on study. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

The animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. 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. The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, open
Vehicle:
other: Acetone/olive oil 4:1
Concentration / amount:
25, 50 and 100 %
Challengeopen allclose all
Route:
other: None, method follows O.E.C.D. test guideline 429.
Vehicle:
other: Acetone/olive oil 4:1
Concentration / amount:
25, 50 and 100 %
No. of animals per dose:
4

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100 %
No. of animals per dose:
4
Details on study design:
The test substance VEHA was formulated in the vehile within two hours of being applied to the mice. 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 mice were treated by daily application of 25 uL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive day.s The test substance 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. The positive control animals were similarly treated to the test animals except that 25 uL of the positive control item, a-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

Five days following the first topical application of the test substance or vehicle all mice were injected via the tail vein with 250 uL of phosphate buffered saline (PBS) containing 3H-methyl thymidine eHTdR: 80 uCi/mL, specific activity 2.0 Ci/mmoL giving a total of 20 uCi to each mouse. Five hours following the administration of 3HTdR all mice weresacrificed 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.

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).

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. 3HTdR incorporation was measured by P-scintillation counting. The number of radioactive disintegrations per minute (DPM) 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)
Statistics:
Group mean

Results and discussion

Positive control results:
a-Hexylcinnamaldehyde, tech., 85% Mean DPM Stimulation Index
25 % v/v 237457 12.5

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Concentration % (v/v) Stimulation Index 0 Vehivle NA 25 1.49 50 3.03 50 5.08 _____________________________________________
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration % (v/v) DPM DPM/Node 0 Vehicle 19023 2378 25 28409 3551 50 57689 7211 100 96629 12079 ______________________________________________

Any other information on results incl. tables

There were no mortalities observed. No clinical signs of systemic toxicity were noted in the test or control animals during the test. The EC3 value of the test substance VEHA was approximately 50 % v/v.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
The test substance Vinyl-2-ethylhexanoate (VEHA) was positive for skin sensitization in the O.E.C.D. test guideline 429 mouse LLNA study. VEHA induced a Stimulation Index of 3-fold at a concentration of 50 % v/v. The EC3 concentration value was 50 % v/v. Classificationand Labeling as a skin sensitizer is required under the GHS and EU CLP.
Executive summary:

The test substance Vinyl-2-ethylhexanoate (VEHA) was assessed for the potential to induce skin sensitization in an O.E.C.D. test guideline 429 mouse Local Lymph Node Assay (LLNA). The test substance VEHA was positive for skin sensitization in the O.E.C.D. test guideline 429 mouse LLNA study. VEHA induced a Stimulation Index of 3-fold at a concentration of 50 % v/v. The EC3 concentration value was approximately 50 % v/v. Classification as a skin sensitizer is required under the GHS and EU CLP.