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Description of key information

Modified Local Lymph Node Assay: moderate skin senistizing (Leidenfrost 2016)

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:
April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
1. non-radioactive alternative, measuring lymph node cell counts 2. in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance
Principles of method if other than guideline:
Modified LLNA (IMDS: Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429. 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 69-75 (2005); Gamer AO et al., Regul. Toxicol. Pharmacol., 52, 290-298 (2008).
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- females nulliparous/ not preganant
- Age at study initiation: 8 weeks
- Weight at study initiation: 28-33 g
- Housing: 1 animal/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 40-70 %
- Air changes (per hr): about 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 2, 20, 100 % test item
No. of animals per dose:
six
Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control (40 % alpha hexyl cinnamic aldehyde) in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2, d3). The volume administered was 25 µl/ear/day. Based on our experiences with the test system and the expected solubility of the test item the following concentrations were used: 0 % (vehicle control), 2 %, 20 % and 100 %. One day after the last application (day 4) the animals were anaesthetized by inhalation of carbon dioxide and sacrificed. The lymphatic organs (the auricular lymph nodes) were removed and transferred into physiological saline (PBS).

INVESTIGATIONS:
- weight of the lymph nodes (given as stimulation index compared to vehicle treated control group)
- cell counts of lymph nodes (given as stimulation index compared to vehicle treated control group; positive if greater or equal as 1.4 stimulation index )
Stimulation indices were calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (positive, if 2 x 10-2 mm increase; 10 % of the control value)
- ear weight (8 mm diameter piece of the right and left ear of each animal by using a punch)
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:
Positive control - sensitizing potential: After treatment with Alpha Hexyl Cinnamic Aldehyde the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals, which are of statistical significance. The "positive level", which is 1.4 for cell count indices, clearly was exceeded (value: 2.21).
Positive control- irritating potential: The "positive level", indicating irritating potential of the test item, which is 1.10 for ear swelling index was exceeded in the positive control group (value: 1.22). This increase was of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was also detected.
Parameter:
SI
Value:
1
Variability:
53.48 %
Test group / Remarks:
vehicle control
Parameter:
SI
Value:
1.19
Variability:
22.62 %
Test group / Remarks:
2 % test substance
Parameter:
SI
Value:
3.3
Variability:
42.14 %
Test group / Remarks:
20 % test substance
Parameter:
SI
Value:
5.03
Variability:
31.83 %
Test group / Remarks:
100 % test substance
Parameter:
SI
Value:
2.21
Variability:
16.58 %
Test group / Remarks:
positive control
Parameter:
other: EC1.4
Value:
3.79
Cellular proliferation data / Observations:
Test item- sensitizing potential: The NMRI mice did show increases in stimulation indices for cell counts and for weights of the draining lymph nodes, which are of statistical significance, after application of the test item at 20 % and 100 % (see table 1, any other information on results incl. tables). In addition, the "positive level", which is 1.4 for cell count indices, was exceeded in these groups (value: 20% = 3.30 and 100% 5.03) (see table 1, any other information on results incl. tables).
Test item- irritating potential: The "positive level", indicating irritating potential of the test item, which is 1.10 for ear swelling index was exceeded in the highest dose group (value: 1.11) (see table 2, any other information on results incl. tables). This increase is of statistical significance. Statistical significant increases for ear swelling were also determined at day 1 in the mid dose group and in the low dose group at day 4. These value are within the normal range. No substance specific effects were determined in any group for ear weights (see table 3, any other information on results incl. tables).

Table 1: Weight index, Cell count index

 Groups  Weight index (index of mean ± SD in %) Cell count index (index of mean ± SD in %)
 Vehicle  1.00 ± 29.32  1.00 ± 53.48
 2 % test item  1.18 ± 19.21  1.19 ± 22.62
 20 % test item  2.03* ± 6.93  3.30* ± 42.14
 100 % test item  4.10* ± 21.38  5.03* ± 31.83
 40 % positive control  1.54* ± 10.42  2.21* ± 16.58

* statistically significant increase (p < 0.05)

Table 2: Ear swelling

 Groups  Day 1 (Ear swelling in mm x10-2) (mean ± SD in %) Day 4 (Ear swelling in mm x10-2) (mean ± SD in %) Index Day 4 
 Vehicle  17.33 ± 2.84  17.75 ± 2.55  1.00
2 % test item  18.00 ± 3.35  18.83* ± 4.98  1.06
20 % test item  18.08* ± 2.85  18.50 ± 4.89  1.04
100 % test item  17.67 ± 4.41  19.75* ± 12.43  1.11
40 % positive control  17.92 ± 4.43  21.67* ± 9.51  1.22

* statistically significant increase (p < 0.05)

Table 3: Ear weight

 Groups  Day 4 (ear weight in mg) (mean ± SD in %)  Index Day 4
   Vehicle  12.95 ± 5.59  1.00
  2 % test item  13.71 ± 4.46  1.06
  20 % test item  12.89 ± 4.73  1.00
  100 % test item  13.81 ± 7.45  1.07
   40 % positive control 18.06* ± 12.47 1.39

* statistically significant increase (p < 0.05)

Interpretation of results:
other: skin sensitizing
Executive summary:

The test item was assessed for both its skin sensitizing and irritant potential in a modified Local Lymph Node Assay with the following concentrations: 0% (vehicle control), 2 %, 20% and 100%. The test item was formulated in acetone / olive oi1 4:1 to yield a solution.

Compared to vehicle treated animals there were increases regarding the cell counts and the weights of the draining lymph nodes in the mid and highest dose group, which are of statistical significance. In addition, the "positive level" of the stimulation index for the cell counts, which is 1.4 was exceeded in these groups. The "positive level", indicating irritating potential of the test item, which is 1.10 for ear swelling index was statistical significant exceeded in the highest dose group . No substance specific effects were determined for ear weights. Therefore there is an indication for a specific skin sensitizing effect after administration of a concentration ~ 20 % test item in this test system. The EC 1.4 value calculated is 3. 79 % for this test item in acetone / olive oil 4: 1. In accordance with the classification proposed in the Technical Report No. 87 of the ECETOC this value corresponds to a moderate skin sensitizer.

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

The test item was assessed for both its skin sensitizing and irritant potential in a modified Local Lymph Node Assay with the following concentrations: 0% (vehicle control), 2 %, 20% and 100%. The test item was formulated in acetone / olive oi1 4:1 to yield a solution.

Compared to vehicle treated animals there were increases regarding the cell counts and the weights of the draining lymph nodes in the mid and highest dose group, which are of statistical significance. In addition, the "positive level" of the stimulation index for the cell counts, which is 1.4 was exceeded in these groups. The "positive level", indicating irritating potential of the test item, which is 1.10 for ear swelling index was statistical significant exceeded in the highest dose group . No substance specific effects were determined for ear weights. Therefore there is an indication for a specific skin sensitizing effect after administration of a concentration ~ 20 % test item in this test system. The EC 1.4 value calculated is 3. 79 % for this test item in acetone / olive oil 4: 1. In accordance with the classification proposed in the Technical Report No. 87 of the ECETOC this value corresponds to a moderate skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the study results, the test substance is classified with Skin Sens. 1B (H317: May cause an allergic skin reaction) according to Regulation (EC) No. 1272/2008 (CLP) as a moderate skin sensitizer.