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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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
Details on test material:
- Name of test material (as cited in study report): vinylacetate
- Physical state: Colourless liquid
- Analytical purity: >99.95%
- Lot/batch No.: Ch 20012003
- Expiration date of the lot/batch: 7 February 2003
- Manufacturer: Wacker-Chemie GmbH, Johannes-Hess Str. 24, D - 84489 Burghausen, Germany
- Storage condition of test material: In the original container in freezer (-20°C±3°C), away from direct sunlight
- Stability: Stable under storage conditions

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL 5960 AD Horst, The Netherlands
- Age at study initiation: 9-13 weeks
- Weight: 16.4-21.4 g (at the beginning of the acclimatisation period)
- Housing: 5 per cage in Makrolon type-3 cages
- Diet: Pelleted standard Kliba 3433, batch #75/02 mouse maintenance diet ad libitum
- Water: Community tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Air changes (per hr): 10-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 29 January 2003 To: 5 February 2003

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5 %, 10 %, 25 %, 50 % (w/v) of vinyl acetate
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in 2 mice with concentrations of 10 %, 25 %, 50 % (w/v) in acetone:olive oil, 4:1 (v/v) and 100 % (undiluted). No irritation effects were observed in the concentrations applied.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 %, 25 %, 50 % (w/v) in acetone:olive oil, 4:1 (v/v) and 100 % (undiluted). The application volume, 25 µL, was spread over the entire dorsal surface (0-8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
- The ear swelling (ear thickness) of both ears (left and right) of mice at application area was measured using calipers prior to the first application, daily 24 (± 2) hours after each dosing and prior to necropsy.
- Five days after the first topical application, all mice were administered with 250 µL of 79.4 µCi/mL 3HTdR (equal to 19.8 µCi 3HTdR) by intravenous injection via a tail vein.
- Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich). Both ears (left and right) of mice at apical area were punched using a biopsy punch (Stietel, 0 8 mm). Both punches of each animal were pooled and immediately weighed using an analytical balance.
- The draining lymph nodes were rapidly excised and pooled for each individual animal (2 nodes per mouse). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OTHER:
- The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBA/CaOlaHsd mice. The validation/positive control study was performed with ALPHA-HEXYLCINNAMALDEHYDE in acetone:olive oil, 4:1 (v/v) using CBA/CaOlaHsd mice (RCC Study Number 846871) between 8-22 January 2003.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations of body weights, ear thickness, ears punches weights and DPM values were calculated.

Results and discussion

Positive control results:
Stimulation indices of 2.5, 3.7 and 9.7 were determined with alpha-hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). The test item alpha-hexylcinnamaldehyde was found to be a sensitizer and an EC3 value of 7.08% (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Stimulation indices of 2.0, 2.4, 1.9, 1.7 and 1.3 were determined with vinyl acetate at 5%, 10%, 25%, 50% (w/v) in acetone:olive oil, 4:1 (v/v) and 100% (undiluted). A Stimulation index of 16.6 was determined with the positive control substance 25% (w/v) in acetone:olive oil, 4:1 (v/v).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
A significant difference of dpm/mouse was determined at concentration of 10% (w/v) comparing with the vehicle control group at p:=0.05 (Dunnett-test, two sides). A significant difference of dpm/mouse was determined between the positive control group and the vehicle control group at p:=0.05 (Dunnett-test, two sides).
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
100%

Any other information on results incl. tables

No test item-related clinical signs were observed in any animals of the vehicle control group or Group 3 (5%). On the second application day a slight to moderate ear swelling was observed at both dosing sites in all mice of Group 2 (HCA, 25%) (moderate), Group 4 (10%) (slight, persisting for two days), Group 5 (25%) (slight), Group 6 (50%) (moderate) and Group 7 (100%, undiluted) (moderate), persisting for the remaining days. All treated animals survived the scheduled study period.

Summary of results

Group

Test substance

% (w/v)

Dpm/Ln M±SD

S.I. M±SD

T value

1 (-ve control)

-

-

161±58

-

-

2 (+ve control)

HCA

25

2677±871

16.6±5.4

6.44**

3

vinylacetate

5

318±74

2.0±0.5

2.24

4

vinylacetate

10

389±217

2.4±1.3

3.26**

5

vinylacetate

25

301±64

1.9±0.4

2.00

6

vinylacetate

50

279±99

1.7±0.6

1.69

7

vinylacetate

100 (undiluted)

208±60

1.3±0.4

0.67

**significant difference at p 5 0.05 (two sides)

The results obtained and reported in the above table, show a negative dose response potential at the concentrations of 25%, 50% and 100% (undiluted), demonstrated by decreasing S.I. values. As some test item related findings such as, slight to moderate ear swelling were observed at the local dosing sites, it might be considered that the effect obtained is based on local irritation rather than on local Langerhans cells as the immuno targets.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Vinyl acetate was non-sensitising when tested up to 100% (undiluted) in the LLNA.
Executive summary:

Five groups each of five female mice were treated with vinyl acetate at concentrations of 5%, 10%, 25%, 50% (w/v) in acetone:olive oil, 4:1 (v/v) and 100 % (undiluted) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A negative control group of five mice was treated with an equivalent volume of the vehicle (acetone:olive oil, 4:1 (v/v)) only. A positive control group of five mice was treated with alpha-hexylcinnamaldehyde at concentration of 25% (w/v) in acetone:olive oil, 4:1 (v/v). The ear swelling (ear thickness) of both ears (left and right) of mice at application area were measured prior to the first application, daily 24 (± 2) hours after each dosing and prior to necropsy. Five days after the first topical application the mice were intravenously injected into a tail vein with radio-labelled thymidine (H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and both ears at apical area were punched and pooled per mouse. The pooled ear punches were immediately weighed. The draining auricular lymph nodes were excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells were determined by the incorporation of 3H-methyl thymidine measured in a R-scintillation counter. No test item-related clinical signs were observed in any animals of the vehicle control group or Group 3 (5%). On the second application day a slight to moderate ear swelling was observed at both dosing sites in all mice of Group 2 (HCA, 25%) (moderate), Group 4 (10%) (slight, persisting for two days), Group 5 (25%) (slight), Group 6 (50%) (moderate) and Group 7 (100%, undiluted) (moderate), persisting for the remaining days. All treated animals survived the scheduled study period.

Vinyl acetate (CAS 108-05-4) was found to be a non-sensitizer when tested up to 100% (undiluted).

Vinyl acetate showed a local irritation at 10%, 25%, 50% (w/v) in acetone:olive oil, 4:1 (v/v) and 100% (undiluted).

The positive control substance showed an allergenic potency when tested at concentration of 25% (w/v) in acetone:olive oil, 4:1 (v/v).