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Administrative data

Description of key information

Local Lymph Node Assay (according to OECD 429; GLP-compliant): not sensitising

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:
Oct 2017
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:
22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the study period has been guaranteed by the sponsor and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in the vehicle was indirectly determined by the concentration control or homogeneity analysis.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparations were prepared on a weight per weight basis shortly before application by stirring with a magnetic stirrer and treatment in an ultrasonic bath. The homogeneity of the test substance preparations during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in propylene glycol; applied as suspension
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V.,Inc., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 10 weeks (pretest); 9 weeks (main test)
- Weight at study initiation: 18.4 g – 20.3 g (pretest); 18.0 g – 21.0 g (main test)
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 26 Sep 2017 To: 18 Oct 2017
Vehicle:
propylene glycol
Concentration:
5%, 10%, 30% (hightest test substance concentration which could be technically prepared)

The highest test substance concentration that does not induce local signs of skin irritation and/or systemic toxicity was determined in a pretest (experimental conduct in accordance with GLP, but without a GLP status).
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed. The highest test substance concentration which could be technically prepared was a 30% test-substance preparation.
- Irritation/ear thickness measurements/erythema scores: After application of a 30% and 5% concentration, the animals did not show relevant signs of local irritation as confirmed by the ear weights (compared to historical vehicle values) and ear thickness measurements. However, the ear skin of the 30% concentration showed well-defined erythema and moderate swelling and the 5% concentration a very slight erythema on study day 2.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.

MAIN STUDY
- Randomization: Prior to first application, the animals were distributed to the individual groups, received their 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“.
- Form of application: epicutaneous
- Application volume: 25 µL per ear
- Side of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- 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.
- 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.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 µCi ³H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice.
- Sacrifice: on study day 5 about 5 hours after ³H-thymidine injection by cervical dislocation under Isoflurane anesthesia
- Terminal procedures:
● Determination of ear weights: 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
6 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 by 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% trichloro-acetic acid (TCA). Each precipitate was
transferred to scintillation fluid and incorporation of ³H-thymidine into the cells was measured in a β-scintillation counter (LSC).


EVALUATION OF RESULTS
- A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of ³H-thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index (SI ≥ 3.0). 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). The thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For the statistical analysis of the the parameters ³H-thymidine incorporation, cell count, lymph node weight and ear weight the Wilcoxon-Test was used.
Parameter:
SI
Remarks:
³H-thymidine incorporation Stimulation Index
Value:
1.38
Test group / Remarks:
5% preparation of the test substance
Parameter:
SI
Remarks:
³H-thymidine incorporation Stimulation Index
Value:
1.61
Test group / Remarks:
10% preparation of the test substance
Parameter:
SI
Remarks:
³H-thymidine incorporation Stimulation Index
Value:
1.03
Test group / Remarks:
30% preparation of the test substance
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
- The test substance did not induce a biologically relevant increase (no increase above the cut off Stimulation Index of ≥ 3) of ³H-thymidine incorporation into the cells from the auricular lymph nodes.
- No biologically relevant response (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) was observed in the auricular lymph node cell counts.
- No relevant increase in lymph node weights was noted at all concentrations.
- No increases (SI ≥ 1.25) in ear weights; demonstrating the absence of relevant ear skin irritation


DETAILS ON STIMULATION INDEX CALCULATION
- Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of ³H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by that of the vehicle control group.

EC3 CALCULATION
- no EC3 was calculated, because no increase above to cut off SI of ≥ 3 was oberved

CLINICAL OBSERVATIONS
- No signs of systemic toxicity
- No local findings


BODY WEIGHTS
- No relevant influence on the mean body weights

In this study ³H-thymidine incorporation was lower as the historic control data. As this effect was observed for all test substance treated groups as well as control group throughout the study, the overall evaluation of the study is not influenced.

Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that the test substance does not exhibit 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 by 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 5%, 10% and 30% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. The 30% preparation was the maximum technically applicable concentration.

The study used 3 test groups and 1 control group. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application, 20 µCi3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring3H-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.

When applied as 30%, 10% and 5% preparation in propylene glycol, the test substance did not induce a biologically relevant increase (no increase above the cut off Stimulation Index of ≥ 3) of3H-thymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, no biologically relevant response (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) was observed in the auricular lymph node cell counts.

In addition, no relevant increase in lymph node weights was noted at all concentrations.

However, statistically significant increases were noted for3H-thymidine incorporation at 5% and for cell count and lymph node weight at 10%.

The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of relevant ear skin irritation.

Thus, it is concluded that the test substance does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

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

The skin sensitizing potential of the test substance was assessed by using the radioactive Murine Local Lymph Node Assay. The assay was conducted according to OECD Guideline 429 under GLP-conditions.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 30% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. The 30% preparation was the maximum technically applicable concentration. The study used 3 test groups and 1 control group. Each test animal was treated with 25 µL per ear of the appropriate test substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application, 20 µCi3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring3H-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.

When applied as 30%, 10% and 5% preparation in propylene glycol, the test substance did not induce a biologically relevant increase (no increase above the cut off Stimulation Index of ≥ 3) of3H-thymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, no biologically relevant response (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) was observed in the auricular lymph node cell counts.

In addition, no relevant increase in lymph node weights was noted at all concentrations.

However, statistically significant increases were noted for3H-thymidine incorporation at 5% and for cell count and lymph node weight at 10%.

The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights, demonstrating the absence of relevant ear skin irritation.

Thus, it is concluded that the test substance does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.