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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 05, 2007 - June 19, 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conform with OECD Guidelines for Testing Chemicals, Updated Guideline 429: Skin Sensitisation: LLNA

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)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Details on test material:
Identity: AES
Batch No.: LSF7
Aggregate state at room temperature: Solid
* Colour: White
Purity: 80.3%
Solubility in water: Very soluble
Stability in water: 30 days at room temperature, in the refrigerator (2 to 8°C) and in the freezer (-20°C)
Storage: In the refrigerator (2 to 8° C), moisture protected
Expiration Date: February 15, 2008
Specific Instructions: Slightly hygroscopic

In vivo test system

Test animals

Details on test animals and environmental conditions:
Test system Mice, CBA/CaOlaHsd
Rationale Recognised as the recommended test system
Source Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands
Number of animals for the pre-test 2 females
Number of animals for the main study 16 females
Number of animals per group 4 females (nulliparous and non-pregnant)
Number of test groups 3
Number of control (vehicle) groups 1
Age 7 -8 weeks (beginning of acclimatisation)
Identification: Single caging. The animals were distributed into the test groups at random and identified by cage
Acclimatisation Under test conditions after health examination. Only animals without any visible signs of illness were
used for the study.
Housing Single
Cage Type Makrolon Type I
Bedding Granulated soft wood bedding
Feed Pelleted standard diet, ad libitum
Water Tap water, ad libitum
Environment Temperature 22°C +/- 3 °C, relative humidity 30-90%, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
2.5, 5 and 10%
No. of animals per dose:
4 (nulliparous and non-pregnant)
Details on study design:
Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test
item concentrations of 2.5, 5, and 10% (w/v) in acetone:olive oil, 4+1 (v/v). The application volume, 25 Sl, was spread over the entire dorsal
surface (~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the
relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare.
Five days after the first topical application, all mice were administered with 250 Sl of 78.1 SCi/ml 3HTdR (corresponds to 19.5 SCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation
through stainless steel gauze (200 Smmesh 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 plastic scintillation vials with 10 ml
of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also
measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of
radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
see table above
Test item concentration % S.I.
Group 2 5 (a) 2.43(b)
Group 3 10 (c) 4.07(d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 6.7% (w/v)

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see table below
other: disintegrations per minute (DPM)
Remarks on result:
other: see table below

Any other information on results incl. tables

Calculation and Results of Individual Data Vehicle: acetone:olive oil, 4+1 (v/v)

Test item concentration % (w/v)  Group  Measurement DPM


 DPM-BG a)


Number of lymph nodes


DPM per lymph nodeb) 
Result S.I.
 ---  BG I  21.08  ---  ----  ---  ---
 ---  BG II  21.33  ---  ---  ---  ---
 ---  1  5181.93  5160.7  8  645.1  ---
 2.5  2  6379.88  6358.7  8  794.8  1.23
 5  3  7687.69  7666.5  8  958.3  1.49
 10  4  5459.49  5438.3  8  679.8  1.05

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all SI´s are below 3.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information
The test item AES was found to be not a skin sensitiser under the described conditions.
Executive summary:

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

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