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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study according OECD 429

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Groups of five mice were treated with the undiluted test material or the test material at concentrations of 25% or 50% v/v in dimethyl formamide. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). A further group of five mice received the vehicle alone in the same manner.
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine ³HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ud) giving a total of 20 µCi
to each mouse.
Five hours following the administration of 3HTdR all mice were killed. For each individual animal of each group the draining auricular lymph
nodes were excised and processed. For each individual anima! 1 ml of PBS was added to the lymph nodes.
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh
stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional
5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive
material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately
450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß-scintillation counting. The "Poly QTM" vials containing the sampIes and scintillation fluid were placed in the sampie changer of the
scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number
of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA).
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Triethyl phosphate
EC Number:
EC Name:
Triethyl phosphate
Cas Number:
Molecular formula:
triethyl phosphate
Details on test material:
content: 99.99%

In vivo test system

Test animals


Study design: in vivo (LLNA)

25%, 50% or undiluted
No. of animals per dose:

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: concentration (% v/v) vehicle 25 50 100 stimulation index N/A 2.53 1.97 2.47
other: disintegrations per minute (DPM)
Remarks on result:
other: concentration (% v/v) vehicle 25 50 100 mean dpm/animal 242 613 478 599

Any other information on results incl. tables

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information
Executive summary:

A study was perfonned to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements ofthe following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004173IEC

Following a preliminary screening test, three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the undiluted test material or the test material as a solution in dimethyl formamide at concentrations of 25% or 50% v/v. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Stimulation index = 2.53 (25% conc.), 1.97 (50% conc.), 2.47 (100% conc.)

Result: negative for all concentrations tested

The test material was considered to be a non-sensitiser under the conditions of the test.