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Description of key information

The skin sensitizing potential was assessed GLP-compliant in a radioactive Murine Local Lymph Node Assay according to OECD Guideline 429. Concentrations of 0.50 % and 1 % led to a SI value (3H-thymidine incorporation) > 3. A significant increase in cell proliferation, cell count, and lymph node weight was observed at all concentrations. The EC3 value was calculated to be 0.12 %. Therefore, the substance is considered to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar - Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landeamt für Umwelt Rheinland-Pfalz
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- Physical state / color: Solid, waxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: good solubility of the preparation was achieved in the vehicle MEK

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparation was produced on a weight per weight (w/w) basis shortly before application. After heating at ca. 40 °C for about 5 minutes and stirring with a magnetic stirrer, the test substance was soluble in the vehicle. Before application, the test substance preparation was cooled down to room temperature.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution
Species:
mouse
Strain:
CBA/Ca
Remarks:
OlaHsd (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553, NL-5800 AN Venray
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.1 g - 22.1 g (pretest), 17.4 g - 22.1 g (main test)
- Housing: The animals were housed in fully air-conditioned rooms in polycarbonate cages type MII with mesh wire tops.
- Diet (e.g. ad libitum): Kliba mouse / rat maintenance diet "GLP" supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Vehicle:
methyl ethyl ketone
Concentration:
0.10 %, 0.50 % nd 1 % preparations of the test substance in methyl ethyl ketone (MEK) or the vehicle alone
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test substance preparations at different concentrations were solutions in methyl ethyl ketone (MEK).
- Irritation: Signs of local irritation were assessed on day 1, 2 and 5.
- Systemic toxicity: No signs of systemic toxicity.
- Ear thickness measurements: Prior to the first application of the test substance (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer.

MAIN STUDY
- Randomization: Prior to the first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions of "Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 - 64".
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and / or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
- Application volume: 25 µL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site
- 3H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 µCi 2H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice.
- Sacrifice: The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
- Determination of ear weight: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 8 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
- Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5 % tricholoro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

EVALUATION OF RESULTS
In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears is determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are lymph node cell count and to a certain extent lymph node weight. Because irritation by the test substance may also induce lymph node responses the weights of ear punches taken from the area of test substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.
A test substance is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test substance results in an incorporation of 3H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or by using all data points.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values and / or single animal values of the test substance treated groups by the mean of the vehicle treated group. Statistical analyses of 3H-thymidine incorporation, cell count, lymph node weight and ear weight were performed according to WILCOXON-Test.
Parameter:
SI
Remarks:
3H-thymidine incorporation Stimulation Index
Value:
2.82
Test group / Remarks:
0.10 % preparation of the test substance
Parameter:
SI
Remarks:
3H-thymidine incorporation Stimulation Index
Value:
5.97
Test group / Remarks:
0.50 % preparation of the test substance
Parameter:
SI
Remarks:
3H-thymidine incorporation Stimulation Index
Value:
9.31
Test group / Remarks:
1 % preparation of the test substance
Parameter:
EC3
Remarks:
3H-thymidine incorporation
Value:
0.12
Test group / Remarks:
calculated by linear regression from the results of the 0.10 % and 0.50 % concentrations
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
When applied as 0.50 % and 1 % preparation in MEK, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.10 % test substance preparation lies just below the cutoff and was statistically significant.
Concomitantly, the 0.50 % and 1 % concentrations induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The SI of the 0.10 % test substance preparation lies just above the border of biological relevance and was statistically significant.
In addition, statistically significant and relevant increases in lymph node weights were noted at all concentrations.
The ear weight stimulation indices did not indicate relevant increases in ear weights (no increase above the cut off Stimulation Index (SI) of ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, the highest concentration (1 %) caused statistically significant and considerably increased ear weights.

DETAILS ON STIMULATION INDEX CALCULATION
Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values and / or single animal values of the test substance treated groups by the mean of the vehicle treated group.

EC3 CALCULATION
The threshold concentration for sensitization induction was around 0.10 % for 3H-thymidine incorporation and cell counts. The EC3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 0.10 % and 0.50 % concentrations to be 0.12 %. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of all concentrations to be 0.10 %.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation.
No local findings were observed during the observation period.

BODY WEIGHTS
No relevant influence on the mean body weights was observed during the study. However, the mean body weight gain was slightly reduced in animals of the 1 % concentration.

Table 1: Stimulation indices

Test group

Treatment

3H-thymidine incorporation

Stimulation Index1

Cell Count

Stimulation Index1

Lymph Node Weight

Stimulation Index1

Ear Weight

Stimulation Index1

1

2

3

4

Vehicle MEK

0.10 % in MEK

0.50 % in MEK

1 % in MEK

1.00

2.82 ##

5.97 ##

9.31 ##

1.00

1.55 ##

2.24 ##

3.02 ##

1.00

1.42 ##

2.13 ##

2.67 ##

1.00

1.05

1.07

1.20 ##

1test group x / test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.05, ## for p ≤ 0.01)

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
It is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.
Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 0.10 %, 0.50 % and 1 % preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone.

Each test animal was treated with 25 µL per ear of the appropriate test substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days.

Three days after the last application, 20 µCi 3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts, and weights of each animal's pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Table 1: Stimulation indices

Test group

Treatment

3H-thymidine incorporation

Stimulation Index1

Cell Count

Stimulation Index1

Lymph Node Weight

Stimulation Index1

Ear Weight

Stimulation Index1

1

2

3

4

Vehicle MEK

0.10 % in MEK

0.50 % in MEK

1 % in MEK

1.00

2.82 ##

5.97 ##

9.31 ##

1.00

1.55 ##

2.24 ##

3.02 ##

1.00

1.42 ##

2.13 ##

2.67 ##

1.00

1.05

1.07

1.20 ##

1test group x / test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.05, ## for p ≤ 0.01)

No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 0.50 % and 1 % preparation in MEK, the test substance induced a biologically relevant (increase to 3 -fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.10 % test substance preparation lies just below the cutoff and was statistically significant.

Concomitantly, the 0.50 % and 1 % concentrations induced a biologically relevant and statistically significant response (increase to 1.5 -fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The SI of the 0.10 % test substance preparation lies just above the border of biological relevance and was statistically significant.

In addition, statistically significant and relevant increases in lymph node weights were noted at all concentrations.

The ear weight stimulation indices did not indicate relevant increases in ear weights (no increase above the cut off Stimulation Index (SI) of ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, the highest concentration (1 %) caused statistically significant and considerably increased ear weights.

Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was around 0.10 %. The EC3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 0.10 % and 0.50 % concentrations to be 0.12 %. The EC1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of all concentrations to be 0.10 %.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 0.10 %, 0.50 % and 1 % preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone.

Each test animal was treated with 25 µL per ear of the appropriate test substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days.

Three days after the last application, 20 µCi 3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts, and weights of each animal's pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Table 1: Stimulation indices

Test group

Treatment

3H-thymidine incorporation

Stimulation Index1

Cell Count

Stimulation Index1

Lymph Node Weight

Stimulation Index1

Ear Weight

Stimulation Index1

1

2

3

4

Vehicle MEK

0.10 % in MEK

0.50 % in MEK

1 % in MEK

1.00

2.82 ##

5.97 ##

9.31 ##

1.00

1.55 ##

2.24 ##

3.02 ##

1.00

1.42 ##

2.13 ##

2.67 ##

1.00

1.05

1.07

1.20 ##

1test group x / test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.05, ## for p ≤ 0.01)

No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 0.50 % and 1 % preparation in MEK, the test substance induced a biologically relevant (increase to 3 -fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.10 % test substance preparation lies just below the cutoff and was statistically significant.

Concomitantly, the 0.50 % and 1 % concentrations induced a biologically relevant and statistically significant response (increase to 1.5 -fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The SI of the 0.10 % test substance preparation lies just above the border of biological relevance and was statistically significant.

In addition, statistically significant and relevant increases in lymph node weights were noted at all concentrations.

The ear weight stimulation indices did not indicate relevant increases in ear weights (no increase above the cut off Stimulation Index (SI) of ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, the highest concentration (1 %) caused statistically significant and considerably increased ear weights.

Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was around 0.10 %. The EC3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 0.10 % and 0.50 % concentrations to be 0.12 %. The EC1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of all concentrations to be 0.10 %.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The stimulation indices in the LLNA (OECD 429) showed a dose-dependent increase. Based on the positive result in the assay leading to an EC3 value of 0.12 %, the substance needs to be classified as a skin sensitizer Cat. 1A according Regulation (EC) No. 1272/2007, as amended for the tenth time in Regulation (EC) No. 2017/776. Additionally, because of the low EC3 value close to the general classification limit for mixtures, an SCL of 0.01 % (applying a safety factor of 10) seems warranted.