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Diss Factsheets

Administrative data

Description of key information

Not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 09 to October 23, 2002
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
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)
Details on test animals and environmental conditions:
- Source:Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.5 g -22.4 g
- Housing: ln groups of four in Makrolon type-3 cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 3433, batch no. 57102 mouse maintenance diet ad libitum.
- Water: Community tap water from ltingen, available ad libitum.

- Temperature (°C): 22 ± 3'C,
- Humidity (%): 30 -70 %
- Air changes (per hr): 10 - 15 air change per hour
- Photoperiod (hrs dark / hrs light): There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

other: ethanol:water, 1:1 (v/v)
1 %, 2.5 %, 5 % and 10 % (w/v)
No. of animals per dose:
groups concentration %(w/v) N° per group
1 control / 4 females
2 2.5 4 females
3 5 4 females
4 10 4 females
Details on study design:
RANGE FINDING TESTS: ln a non-GLP conform pretest in two mice, test item concentrations ol 1%, 2.5 %, 5% and 10 % were tested on one ear each. No irritation effects were observed at the concentrations applied.
10 % (w/v) was the highest technically applicable concentration that could sufficiently be dosed to achieve optimal skin contact.

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% (wlv) in ethanol:water, 1:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (Ø - 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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

H-methyl thymidine (HTdR) was purchased from Amersham lnternational (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 82.49 µCi/mltHTdR (equalto 20.6 µCi HTdR) by intravenous injection via a tail vein.

Approximately five hours after treatment with HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zürich).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymphnode cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing three 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 about 42 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of HTdR incorporation was then measured on a p-scintillation counter. Similarly, background HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
The p-scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

ln addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Twice daily from acclimatization start to the termination of in-life phase.
Body weights: At acclimatization start and prior to necropsy.
Clinical signs (local/ systemic): Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
ca. 1.8
Test group / Remarks:
Group 2
Remarks on result:
other: Test item concentration 2.5 % (w/v)
ca. 1.5
Test group / Remarks:
Group 3
Remarks on result:
other: Test item concentration 5 % (w/v)
ca. 1.7
Test group / Remarks:
Group 4
Remarks on result:
other: Test item concentration 10 % (w/v)


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 at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
other: CLP criteria not met
Non sensitising
Executive summary:


The allergenic potential of the test substance was assessed according to the OECD guideline 429. Three groups of four female mice were used, each treated with the test item at concentrations of 2.5, 5 and 10 % (w/v) in ethanol:water, 1:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 1:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a pscintillation counter.


No test item-related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of "HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

Test item concentration       %(w/v)        S.I

Group 2                                  2.5         1.8

Group 3                                  5           1.5

Group 4                                  10           1.7

An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.l. value of 3.



The test item was found to be a non sensitizer when tested up to 10 % (wlv) in ethanol:water, 1:1 (v/v).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No studies on the "Skin sensitisation" are available for the substance in itself.

Nevertheless a study was conducted with an analogue molecule (Similar Substance 03). Further information are reported in the Read Across justification attached to section 13.

The study was performed according to the OECD guideline 406 and EU method B.6.

Ten males were used as control group and 20 males were used as test group.

The highest non-irritating test article concentration used for challenge application was 10 % in vaselinum album.

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

In this study 0 % of the animals were positive after treatment with the highest non-irritant test substance concentration of 10 % in vaselinum album. Therefore the test article is considered to be a non sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification


Category 1

Substances shall be classified as skin sensitizers in category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

Specific criteria of animal test:

when an adjuvant type test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

For a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive.

Furthermore, stimulation index of three or more is considered a positive response in the local lymph node assay.

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test-≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

Based on animal test (Local lymph node assay) results performed, according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the test substance is Not Classified as the Stimulation Index is below the threshold of classification at all concentrations.