Registration Dossier

Administrative data

Description of key information

A LLNA in mice showed no skin sensitizing potential.

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:
2015
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:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
Deviations:
yes
Remarks:
Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Stability under test conditions: The formulations of the test item in the vehicle were visually described as solutions. Stability of the test item in the vehicle was analytically verified for up to 4 days. A content check confirmed the formulations within the defined limits.
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Crl:NMRI BR
- females: nulliparous and not pregnant
- Age at study initiation: 8 weeks
- Weight at study initiation: 28 - 32 g
- Housing: during adaption 8 mice were housed together; during the study period mice were single-housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): about 10 per hour
- Photoperiod (hrs dark / hrs light): 12 h rhythm
- IN-LIFE DATES: November 2015
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
The formulations of the test item in the vehicle were visually described as solutions. Stability of the test item in the vehicle was analytically verified for up to 4 days. A content check confirmed the formulations within the defined limits.
Concentration:
0 (vehicle control), 2 % in vehicle, 20 % in vehicle, 100 % (pure test item; no vehicle)
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item (as formulation in vehicle or pure) or the vehicle was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
The so-called stimulation (or LLN-) index is calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is about 1.00 and the indices of vehicle treated animals are set to 1.00.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- body weights
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The individual values from actively treated groups were compared with those from the control group. A pre-testing was carried out by a Cochran test Furthermore, depending on the statistical result, a Dunnett test significance test was conducted (significance levels of 5 %; two-tailed).
In this method of statistical processing of measurements a large number of comparisons are made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used for the final evaluation of the biological relevance.
Positive control results:
Alpha hexyl cinnamic aldehyde in a concentration of 40 % in acetone/olive oil shows a clear sensitising potential in the local lymph node assay (IMDS). Weight and cell count index in the lymph nodes as well as ear swelling and ear weight were clearly and statistically significant increased.
Parameter:
SI
Remarks:
Stimulation Index based on cell count in lymph nodes (compared to negative control level)
Value:
1.17
Test group / Remarks:
100 % (pure test item)
Remarks on result:
other: Cell count index in draining lymph nodes: 'positive level' of 1.4 (cut-off for skin sensitization) was not exceeded in any dose group

Results of LLNA (means for 6 animals per group)

 

Parameters investigated

Vehicle

Low dose (2 % in AO)

Medium dose (20 % in AO)

High dose (100 %)

 positive control HCA (40 % in AO)

Stimulation Index: weight of draining lymph nodes

 1.00

 0.96

 0.88

 1.16

 1.77*
 Stimulation index: cell count in draining lymph nodes   1.00   0.96 0.82   1.17  1.68*
 Ear swelling in mm x 10E-2 (index), day 4   17.75 (1.00) 18.08 (1.02) 18.92 (1.07)   24.75* (1.39)  21.17* (1.17)
 Ear weight in mg (index)   12.43 (1.00)   12.62 (1.02) 13.59 (1.09) 15.33* (1.23)  14.97* (1.19)

* statistically significant increase (p<= 0.05)

Body weights were not affected by treatment.

A LLNA/IMDS was carried out in female NMRI mice with epicutaneous application of a formulation containing 2 or 20 % of the test item in acetone/olive oil (AO) or the pure test item (100 %) for 3 consecutive days onto both ears of the animals. The results of this study do not point towards a specific immuno stimulating (sensitizing) potential of the test item. After treatment there was no relevant increase compared to control animals (vehicle treated) regarding the cell counts of the draining lymph nodes at all doses. The "positive level", which is 1.4 for cell counts, has not been exceeded in any dose groups, including the pure test item.

Therefore, the concentration of 100 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

A statistically significant increase in ear swelling and ear weight was observed for the neat test item, pointing to an acute irritant (inflammatory) response.

Interpretation of results:
other: negative for skin sensitization
Executive summary:

The test substance (40 % solution in ethyl acetate) was investigated for both its skin sensitizing and irritant potential in the modified Local Lymph Node Assay (LLNA) on female mice, performed according to OECD TG 429. Concentrations of 0 (vehicle control), 2 and 20 % test substance in acetone/olive oil and the pure test substance were tested. Body weights were not affected by treatment. Compared to vehicle control no relevant increase of cell counts in the draining lymph nodes was noted for the test substance at all concentrations. Thus, the test substance should be considered as no inducer of skin sensitization under the conditions of this test. An increase in ear swelling and ear weight point to an acute irritant (inflammatory) response.

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

The technical form of the registered substance is a solution in ethyl acetate (ca. 40 % w/w). The solvent free substance solidifies as a glassy but still sticky material. It is not possible to obtain a powdered sample in a mortar for testing and thus, testing of the isolated (pure) registered substance for skin and eye irritation and skin sensitization is technically not feasible. Therefore, and since the solution is the only form humans can come into skin/eye contact with, it was decided to apply the technical product for these toxicological tests. The 'test item' is thus the 40 % solution in ethyl acetate.

According to data of registrants published on the ECHA homepage the solvent ethyl acetate (CAS-No. 141-78-6) is not a skin sensitizer.

The test item was investigated for both its skin sensitizing and irritant potential in the modified Local Lymph Node Assay (LLNA) on female mice, performed according to OECD TG 429. Concentrations of 0 (vehicle control), 2 and 20 % test item in acetone/olive oil and the pure test item were tested. Body weights were not affected by treatment. Compared to vehicle control no relevant increase of cell counts in the draining lymph nodes was noted for the test item at all concentrations. Thus, the test item and as such the registered substance should be considered as no inducer of skin sensitization under the conditions of this test. An increase in ear swelling and ear weight point to an acute irritant (inflammatory) response.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the negative outcome of a LLNA performed in 2015 no classification is warranted for skin sensitization.

No data are available for respiratory sensitization.