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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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material


In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at start of treatment: 1st pre-test: 9-10 weeks; 2nd pre-test: 10-11 weeks; main study: 8-9 weeks
- Weight at study initiation: 18.3 - 21.7 g
- Housing: group housing in Makrolon cages Type II (pre-test), III (main study), with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Bedding: Granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water (e.g. ad libitum): Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

- Temperature (°C): 22 ± 2
- Humidity (%): 45 – 65% (acclimation period and pre-tests); 32 – 65% (main study)
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

methyl ethyl ketone
0, 2.5, 5, 10% (w/w)
No. of animals per dose:
Details on study design:
- Compound solubility: The highest test item concentration, which could be technically used was the undiluted test item (100%). Test item solution at different concentrations could be prepared using the vehicle methyl ethyl ketone. Vortexing and warming to about 37°C were used to formulate the test item.
- Irritation: At the tested concentrations 50 and 100% (w/w) the animals did not show any signs of systemic toxicity. From day 2 to day 6, both treated animals showed an erythema of the ear skin (Score 3 at a maximum at both test item concentrations). Furthermore, a visible swelling of the ears was observed in the animal treated with 50% test item concentration on day 3, and in the animal treated with the undiluted test item on day 6.
Thus, a second pre-test was performed using test item concentrations of 10 and 25% (w/w). From day 2 to 6, the animal treated with 25% test item concentration showed an erythema of the ear skin (Score 3 at a maximum). Furthermore, slight scurf formation was observed in this animal on day 6 only. The animal treated with 10% test item concentration showed an erythema on day 2 and 3 (Score 2 at a maximum).
Based on these results, the test item in the main study was assayed at 2.5, 5, and 10% (w/w).
- Lymph node proliferation response: not determined

Test Item Preparation:
The test item was placed into an appropriate container on a tared balance and methyl ethyl ketone was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in methyl ethyl ketone. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø~8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

Administration of ³H-Methyl Thymidine:
³H-methyl thymidine (³HTdR) was purchased from Hartmann Analytics, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.7 µCi of ³HTdR (equivalent to approximately 78.8 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.

Determination of Incorporated ³HTdR:
Approximately five hours after treatment with ³HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium (Release, WDT, 30827 Garbsen, Germany). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh 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 (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Determination of Lymph Node Weight and Cell Count:
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY®1, Schärfe System, Reutlingen, Germany). The values obtained were taken down manually.

Determination of Ear Weights:
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Interpretation of Raw Data:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of ³HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response and the cutoff-value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cutoff-values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.
Furthermore, according to OECD guideline 429, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation.

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: At least once daily from experimental start to necropsy.
- Body weights: In the pre-tests: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with ³HTdR.
- Ear thickness: In the pre-tests prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).
- Ear weights: In the pre-tests and main experiment after sacrifice; biopsy punches were taken from each ear.
- Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
- Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
- Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: Concentration % (w/w); Stimulation Index 0%; 1.00 2.5%; 3.42 5.0%; 8.41 10.0%; 14.20
other: disintegrations per minute (DPM)
Remarks on result:
other: Concentration % (w/w); Mean DPM per animal 0 %; 533.1 2.5 %; 1822.3 5.0 %; 4484.5 10.0 %; 7569.5

Any other information on results incl. tables

Viability / Mortality:

No deaths occurred during the study period.

Clinical Signs:

No signs of systemic toxicity were observed during the study period. On application day 2 and 3, the animals treated with test item concentrations of 5 and 10% showed an erythema of the ear skin (Score 1). On day 4, only the animals of the high dose group showed an erythema of the ear skin (Score 1).

Body Weights:

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.

Lymph Node Weights and Cell Counts:

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weight and –cell count was obtained in all test item treated groups in comparison to the

vehicle control group (p<0.05) and a clear dose response was observed. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in all dose groups (indices of 2.08, 3.55, and 4.94).

Ear Weights:

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cutoffvalue

of 1.1 was reported for a positive response of the ear weight index regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU