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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:
26 Oct 2005 - 15 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK Limited, UK
- Strain: CBA/Ca/Ola/Hsd
- Diet (e.g. ad libitum): Diet (RM1), supplied by Special Diet Services Limited, UK
- Water (e.g. ad libitum): Mains water
- Acclimation period: 5 days


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


Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 2.5, 5, 10 or 25 % 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
Groups of four female mice were used for the main LLNA study. Approximately 25μl of a 1, 2.5, 5, 10 or 25 % 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 20 μCi of a 2.0 Ci/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 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 1ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant
(Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

Clinical observations: Animals were checked at least once daily for signs of systemic toxicity.
Bodyweights: 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 study: Approximately 25μl of a 5%, 10% or 25% w/v preparation of hexylcinnamaldehyde in acetone in olive oil was applied, and a vehicle control group was similarly treated using acetone in olive oil alone (4:1).

Data evaluation:
The results are expressed as disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio known as the stimulation index (SI), for each concentration. 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.

EC3 calculations
The estimated concentration of the test substance required to produce a 3-fold increase in the draining lymph node cell proliferative activity was calculated (EC3).
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
The quantity applied per square centimetre was derived from this value, assuming that the area of the mouse ear is 1cm2 and that 1μl is equivalent to 1mg.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone in 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: 8.7
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: At 25 % w/v 51892 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

5964

746

N/A

1

8

4722

590

0.8

2.5

8

7956

995

1.3

5

8

8213

1027

1.4

10

8

15161

1895

2.5

25

8

51892

6487

8.7

EC3

Calculated to be 11.2 % (2800 µg/cm2)

N/A- not applicable

The application of the test substance at concentrations of 1, 2.5, 5, 10 and 25 % w/v in acetone in olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3-fold at the 25 % 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 11.2 % w/v (2800 μg/cm2), indicative of a sensitiser of weak potency.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information