Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication

Data source

Reference
Reference Type:
publication
Title:
Ethanol and Diethyl Phthalate: Vehicle Effects in the Local Lymph Node Assay
Author:
Lalko J, Isola D, and Api AM
Year:
2004
Bibliographic source:
International Journal of Toxicology 23: 171-177

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
acc. Kimber et al. (1992, 1994).
GLP compliance:
not specified
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): hydroxycitronellal
- Supplier: Bush Boake Allen Limited, UK
- Analytical purity: 98.7 %

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17 - 21 g
- Housing: groups of 4 per cage
- Diet (e.g. ad libitum): ad libitum, Porton combined Diet, pelleted diet; Special Diets Services Ltd., Witham, UK
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: Diethyl phtalate (DEP); 1:3 Ethanol:DEP; 3:1 Ethanol:DEP; Ethanol
Concentration:
0%, 1%, 3%, 10%, 30%, 50%
No. of animals per dose:
4
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A material was considered a sensitizer if at least one concentration of the test material was observed to have an stimulation index (SI) value of 3 or more. The EC3 value, or estimated concentration of test material required to elicit an SI of 3 or more, was derived from the dose-response data by linear interpolation. EC3 value was calculated utilizing the equation presented by Basketter et al. (1999)


TREATMENT PREPARATION AND ADMINISTRATION:
Groups of 4 male mice were dosed at one of five concentrations in one of four vehicles or to the same volume of the vehicle alone, which acted as the control. Dosing occurred daily for 3 consecutive days.
The animals "rested" for 2 days and on the 6th day after the first application, all mice were injected intravenously by the tail vein with 250 µl of phosphate-buffered saline (PSB) containing 20 µCi of [3H] methyl thymidine (3HTdR; specific activity 2.0 Ci/mmol).
Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled for each experimental group.
Suspensions of the lymph node cells were prepared by mechanical disaggregation through 200 mesh stainless steel gauze.
The cell suspensions were washed three times with PBS and precipitated overnight at 4 C with 5% w/v trichloroacetic acid (TCA).
The samples were then pelleted by centrifugation. The cells were resuspended in 1 ml of TCA and transferred to scintillation vials containing 10 ml of scintillation fluid. The incorporation of 3HTdR was measured by beta-scintillation counting expressed as counts per minute (cpm) per lymph node for each experimental group.
For each concentration of test material, a stimulation index (SI) relative to the concurrent vehicle-treated control was calculated.
The SI value for each test material was calculated by dividing the mean cpm at a given dose level by the mean cpm of the vehicle control group.
Statistics:
SI values greater than or equal to 3 are considered to result in a positive response. The SI is calculated by dividing the mean cpm at a given dose level by the mean cpm of the vehicle control group.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI (0%, 1%, 3%, 10%, 30%, 50%): - DEP: na, 1.3, 1.5, 1.8, 4.2, 5.3 - 1:3 Ethanol:DEP: na, 1.1, 0.9, 2.1, 4.0, 6.2 - 3:1 Ethanol:DEP: na,1.6, 1.6, 1.4, 4.0, 5.8 - Ethanol: na, 1.3, 1.0, 1.6, 3.3, 7.2
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Hydroxycitronellal showed in all four vehicles a positive response at 30 and 50 % (w/v). DPM (0%, 1%, 3%, 10%, 30%, 50%): - DEP: 267, 345, 391, 495, 1128, 1405 - 1:3 Ethanol:DEP: 241, 275, 225, 507, 971, 1491 - 3:1 Ethanol:DEP: 145, 232, 234, 207, 582, 843 - Ethanol: 175, 223, 181, 276, 579, 1264

Any other information on results incl. tables

The sensitization potential of the test material hydroxycitronellal was greatest 
 when the vehicle was 1:3 Ethanol:DEP. 
The strength of the sensitization response  was observed to vary with the vehicle.

Derived EC3 values:  
1. diethyl phthalate (DEP): 19.7%
2. ethanol:diethyl phthalate 3:1: 22.2%
3. ethanol:diethyl phthalate 1:3: 19.3%
4. ethanol: 26.4%

Applicant's summary and conclusion