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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:
supporting study
Study period:
25 Oct 2005 - 22 Nov 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted in compliance with GLP regulations

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006
Reference Type:
publication
Title:
Comparative analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl acrylate, and ethylhexyl acrylate) using the local lymph node assay.
Author:
Dearman RJ, Betts CJ, Farr C, McLaughlin J, Berdasco N, Wiench K, Kimber I
Year:
2007
Bibliographic source:
Contact Dermatitis 57: 242-247

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
2-ethylhexyl acrylate
EC Number:
203-080-7
EC Name:
2-ethylhexyl acrylate
Details on test material:
- Name of test material (as cited in study report): ethylhexyl acrylate
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: 99.84 %
- Purity test date: 10 Feb 2006 (reanalysis)
- Lot/batch No.: CTL bottle No 281914, 011093eda0, GV/T No. 00/0903-3
- Expiration date of the lot/batch: 31 May 2006 (reanalysed February 2006)
- Stability under test conditions: stable
- Storage condition of test material: Ambient temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca/Ola/Hsd
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: Young adults (8-12 weeks of age)
- Mean body weight at study initiation: 19.2 g
- Housing: maximum of 4 mice per cage
- Diet (ad libitum): Diet (RM1), supplied by Special Diet Services Limited, Witham, Essex, UK
- Water (ad libitum): mains water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5, 1, 2.5, 5 and 10 % w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS
A preliminary sighting study was conducted on one animal per dose group for 2.5, 10 and 50 % w/v concentrations to determine the acceptable toxicity and lymph node activation levels.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of the test substance
should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.


TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four female mice were used for the main LLNA study. Approximately 25μl of a 0.5, 1, 2.5, 5 or 10 % w/v preparation of the test substance in acetone in olive oil (4:1) was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. The procedure was repeated daily for 3 consecutive days.

Three days after the third application, all the animals were injected, via the tail vein, with approximately 250μl of phosphate buffered saline (PBS) containing 20μCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10mL of PBS. Approximately 3mL of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA.

The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Animals were checked at least once daily for signs of systemic toxicity.

The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3H-methyl thymidine on day 6 for LLNA study mice or prior to termination for sighting study mice.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was derived by interpolating between two points on the Stimulation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle-treated control values [SI=1] not being used for the latter). Where the data points lying immediately above and below the SI value of three have the co-ordinates a (the concentration giving the SI immediately above 3), b (the SI of a), c (the concentration giving the SI immediately below 3) and d (the SI of c), the EC3 value was calculated using the following equation:
EC3 = [(3-d)/(b-d)] x (a-c) + c

Results and discussion

Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 % w/v in acetone : olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 25 % w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 3.1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: At 10 % w/v 17989 dpm.

Any other information on results incl. tables

Concentration of

test substance

(% w/v)

Number of lymph

nodes assayed

Disintegrations

per minute

(dpm)

dpm per

lymph node

Test : control

ratio (SI)

0 (vehicle only)

8

5822

728

N/A

0.5

8

6172

772

1.1

1

8

7026

878

1.2

2.5

8

5503

688

1.0

5

8

7012

877

1.2

10

8

17989

2249

3.1

EC3

Calculated to be 9.7 % (2425μg/cm2)

N/A - not applicable

The application of the test substance at concentrations of 0.5, 1, 2.5, 5 and 10 %w/v in acetone : olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3-fold at the 10 % w/v concentration. Consequently, the test substance was shown to be a potential skin sensitiser. The concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated to be 9.7 % w/v (2425 μg/cm2), indicative of a sensitiser of moderate potency.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
According to test results, the test substance is a sensitiser of moderate potency.