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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
Study period:
05 - 20 Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 Apr 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Directive No. 2004/73/EC
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyl benzoate
EC Number:
212-214-3
EC Name:
Vinyl benzoate
Cas Number:
769-78-8
Molecular formula:
C9H8O2
IUPAC Name:
vinyl benzoate

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
(CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited, Bicester, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood flakes
- Diet: Global Rodent diet 2014 (Harlan Teklad, Blackthorn, Bicester, Oxon, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
preliminary study: 100%
main study: 100, 50 and 25% (v/v)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TEST:
- Compound solubility: Vehicle was chosen as it produced the most suitable formulation (solution) at the required concentration in a solubility test.
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the undiluted test material 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
- Irritation: No excessive local irritation.
- Systemic toxicity: No signs of systemic toxicity were noted.

MAIN STUDY: Based on the preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% v/v, this concentration was selected as the highest dose for the main test. In addition, 25 and 50% dosing groups and vehicle group were included in the main study. Positive control was tested separately from 11 - 17 Ap 2008 in five animals under same conditions as the main test.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine determined by β-scintillation counting
- Criteria used to consider a positive response: The test substance will be regarded as a sensitiser if at least one concentration results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. Negative control animals were dosed with the vehicle, acetone/olive oil solution. Five days after the third application on Day 3, an injection in the tail vein of 0.25 mL (20 µCi to each mouse) of 3HTdR solution (80 µCi/mL) was made. Approximately 5 h after 3HTdR injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal. A single cell suspension was prepared by separation through a stainless steel gauze. After 2 centrifugation steps, the cells were resuspended in 5% trichloroacetic acid (TCA) and incubated for approx. 18 h to precipitate out the radioactive material. After centrifugation cells were resuspended in 1 mL of TCA and 10 mL of scintillation fluid. 3HTdR was measured by β-scintillation using a scintillation system.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:
The SI for current positive control (α-hexylcinnamaldehyde 15% (v/v) in acetone/olive oil 4:1) was 10.91.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
4.19
Test group / Remarks:
25% (v/v)
Key result
Parameter:
SI
Value:
8.48
Test group / Remarks:
50% (v/v)
Key result
Parameter:
SI
Value:
14.13
Test group / Remarks:
100%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

EC3 CALCULATION: No EC3 value was given in the study report.

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
other: Skin Sens Cat 1 according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 3 at concentrations of 25, 50 and 100%. Therefore, the test substance is considered to be a skin sensitiser.