2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 9th of September 2003 to the 28th November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: August 2005 Date of signature: May 2002

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-690-9

EC Name:

-

Cas Number:

86273-46-3

Molecular formula:

C9H14O4

IUPAC Name:

2-[2-(ethenyloxy)ethoxy]ethyl prop-2-enoate

Test material form:

other: liquid

Details on test material:

Identity: 2-(2'-Vinyloxy ethoxy)ethyl acrylate
Description: Colourless liquid
Batch number: 2C02-3323-A2
Purity: 99.6 %
Stability of test item: Stable under storage conditions
Expiry date: 31-JAN-2004
Storage conditions: in the original container in refrigerator (5"C ± 3 "C), away from direct sunlight.
Safety precautions: Routine hygienic procedures (gloves, goggles, face
mask).

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) were supplied by Harlan Netherlands, BV. Postbus 6174, NL - 5960 AD Horst / The Netherlands


- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 16 g - 24 g

- Housing:
In groups of four in Makrolon type-3 cages with standard softwood bedding

- Diet (e.g. ad libitum):
ad libitum

- Water (e.g. ad libitum):
ad libitum.

- Acclimation period:
At least five days. Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
ENVIRONMENTAL CONDITIONS

- Temperature (°C):
The temperature was controlled to remain within the target ranges of 19 to 25 deg C.

- Humidity (%):
The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr):
The rate of air exchange was between 10 and 15 changes per hour.

- Photoperiod (hrs dark / hrs light):
There was a 12 hour fluorescent light 1 12 hour dark cycle with at least 8 hours music during the light period.

IN-LIFE DATES:
From: Day 0 To: End of study

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Main test: 5%, 10%, 25% (w/v) in acetone:olive oil, 4:1 (v/v)

No. of animals per dose:

4

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 two mice with concentrations of 100/0, 25 %, 50 % (w/v) and 100 % (undiluted) (pretest excluded from Statement of Compliance).

The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

TREATMENT PREPARATION AND ADMINISTRATION:
Five days after the first topical application, all mice were administered with 250 µI of 81.38 µCi/mI 3HTdR (equal to 20.3 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR:
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich).

The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 ~m 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 f:l-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION OF RAW DATA
REPORT
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM!node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM!node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None

Results and discussion

Positive control results:

Alpha-Hexylcinnamaldehyde, was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.3

Test group / Remarks:

5% (w/v) test item

Results 2

Parameter:

SI

Value:

9.8

Test group / Remarks:

10% (w/v) test item

Results 3

Parameter:

SI

Value:

6.9

Test group / Remarks:

25% (w/v) test item

Any other information on results incl. tables

Calculations and results of individual data: The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3 H-methyl thymidine measured on a ß-scintillation counter.

	Test item concentration % (w/v)	S.I.
Group 2	5	4.3
Group 3	10	9.8
Group 4	25	6.7

An exact EC3 value could not be calculated because this calculation requires data lying immediately aboce and below S.I. values of 3.

Mortality: No deaths occurred during the study period.

Bodyweight: The body weight of the animals, recorded prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Clinical signs: No test item-related clinical signs were observed in any animals of the control group or Group 2 (5 %). On the second and the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (25 %) and Group 3 (10%), respectively, persisting for the remainder of the in-life phase of the study.

Positive Control:

No test item-related clinical signs were observed in any animals of the control group, Group 2 (5 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for four days. On the third application day, a slight ear swelling was also observed at both dosing sites in all mice of Group 4 (25 %), persisting for the remainder of the in-life phase of the study.

All treated animals survived the scheduled study period.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation index (S.I.). The estimated concentration of test item required to produce a S.1. of 3 is referred to as the EC3 value.

Mortality:

No deaths occurred during the study period

Clinical signs:

No test item-related clinical signs were observed in any animals of the control group, Group 2 (5 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for four days. On the third application day, a slight ear swelling was also observed at both dosing sites in all mice of Group 4 (25 %), perSisting for the remainder of the in-life phase of the study.

Body weights:

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

	Tets item concentration % (w/v)	S.I.
Group 2	5	1.5
Group 3	10	3.2
Group 4	25	6.9
EC3 = 9.4 % (w/v)

In this study stimulation indicies of 1.5, 3.2 and 6.9 were determined with the test item at concentrations of 5 %,10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the stimulation index (S.I.).

The positive control item Alpha-Hexylcinnamaldehyde was found to be a skin sensitizer and an EC3 value of 9.4 % (w/v) was derived.

Applicant's summary and conclusion

Interpretation of results:

other: sensitising

Conclusions:

In this study Stimulation indices of 4.3, 9.8 and 6.7 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared. With concurrent controls, as indicated by the Stimulation index (S.I.). The test item 2-(2'-Vinyloxy ethoxy) ethyl acrylate was found to be a skin sensitizer.

Executive summary:

In order to study a possible contact allergenic potential of 2-(2'-Vinyloxy ethoxy)ethyl acrylate, three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4: 1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle acetone:olive oil, 4: 1 (v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. 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 was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

No test item-related clinical signs were observed in any animals of the control group or Group 2 (5 %). On the second and the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (25 %) and Group 3 (10 %), respectively, persisting for the remainder of the in-life phase of the study. All treated animals survived the scheduled study period.

In this study Stimulation indices of 4.3, 9.B and 6.7 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation index (S.I.). The test item 2-(2'-Vinyloxy ethoxy)ethyl acrylate was found to be a skin sensitizer.