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Description of key information

The test substance has no skin senitizing potential. (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:
Nov. 2015
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:
modifications: 1. non-radioactive alternative, measuring lymph node cell counts; 2. in addition, measurement of ear swelling and ear weight 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
- Age at study initiation: 8 weeks
- Weight at study initiation: 28-34 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:
dimethylformamide
Concentration:
0, 2, 10, 30 %
No. of animals per dose:
6
Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control (30 % 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 %, 10 % and 30 %. A preparation of a formulation >30 % was not possible. 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 Bonferroni-Holm test (Mann-Whitney test included) or 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. The positive level
which is 1.4 for cell count indices, clearly was exceeded.
Positive control - irritating potential: The positive level of ear swelling, which is 2x10-2 mm increase, i.e. about 10 % of the control values, was exceeded in the positive control group. This increase is of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was also detected.
Parameter:
SI
Remarks on result:
other: see cellular proliferation and Any other information on results incl. tables
Cellular proliferation data / Observations:
The NMRI mice did not show increases in stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item. The positive level indicating sensitizing potential, which is 1.4 for the cell count index, was never reached or exceeded in any dose group (see Table 1). The positive level of ear swelling indicating irritating potential, which is 2 x 10-2 mm increase, i.e. about 10 % of the control values, was not reached or exceeded in any dose group (See Table 2).

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 ± 25.49  1.00 ± 29.31
 2 % test item  1.19 ± 24.33  1.12 ± 28.75
 10 % test item  1.12 ± 16.00  1.01 ± 25.16
 30 % test item  1.61 ± 44.76  1.33 ± 52.79
 30 % positive control  1.49* ± 31.06  1.94* ± 37.11

* 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  17.33 ± 2.84  17.58 ± 2.93  0.99
10 % test item  17.33 ± 2.84  17.50 ± 3.85  0.99
30 % test item  17.17 ± 3.36  17.92 ± 4.43  1.01
30 % positive control  17.83 ± 3.24  26.08* ± 13.53  1.47

* statistically significant increase (p < 0.05)

Table 3: Ear weight

 Groups  Day 4(ear weight in mg)(mean ± SD in %)  Index Day 4
   Vehicle  12.97 ± 4.27  1.00
  2 % test item  12.48 ± 3.40  0.96
  10 % test item  12.00 ± 3.41  0.93
  30 % test item  12.10 ± 4.47  0.93
   30 % positive control  20.18* ± 9.51 1.56

* statistically significant increase (p < 0.05)

Only after treatment with the positive control 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 (table 1). The positive level, which is 1.4 for cell count indices, clearly was exceeded.

In addition, ear swelling and the ear weight were only increased after treatment with the positive control (table 2, table 3).

Interpretation of results:
other: no indication for a specific skin sensitizing effect
Remarks:
in opinion of study director
Executive summary:

The test item was investigated in the modified local lymph node assay (LLNA-IMDS) on female NMRI mice according to OECD 429. Concentrations of 0 (vehicle control), 2, 10 or 30 % formulated in dimethylformamide were tested. Validity of the assay was demonstrated by the positive results obtained with positive control compound alpha hexyl cinnamic aldehyde.

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test item.

Taken together, no antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 30 % test item by the method used. Therefore, the concentration of 30 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

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

The test item was investigated in the modified local lymph node assay (LLNA-IMDS) on female NMRI mice according to OECD 429. Concentrations of 0 (vehicle control), 2, 10 or 30 % formulated in dimethylformamide were tested. Validity of the assay was demonstrated by the positive results obtained with positive control compound alpha hexyl cinnamic aldehyde.

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test item.

Taken together, no antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 30 % test item by the method used. Therefore, the concentration of 30 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.



Justification for selection of skin sensitisation endpoint:
Only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the study results, a classification according Regulation (EC) No. 1272/2008 (CLP) is not warranted.

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