Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). The IMDS was validated and published with scientific justification 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:
IDMS LLNA: 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.
Principles of method if other than guideline:
This study is performed according to OECD TG 429. As stated in OECD TG 429 besides the classical radioactive method ‘other endpoints for assessment of the number of proliferating cells may be employed’ as so-called ‘me-too’ tests, if the required performance standards are fulfilled, they are ‘based on similar scientific principles and measure or predict the same biological or toxic effect’ and they are validated.
Here, an alternative method is used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes (IMDS LLNA; Integrated Model for the Differentiation of Skin Reactions). In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitizing properties. Information on validation of the IMDS LLNA 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).
In the IMDS LLNA stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify skin sensitization) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
13-β-ethyl-3-methoxygona-2,5(10)-dien-17-one
EC Number:
219-034-4
EC Name:
13-β-ethyl-3-methoxygona-2,5(10)-dien-17-one
Cas Number:
2322-77-2
Molecular formula:
C20 H28 O2
IUPAC Name:
3-Methoxy-18-methyl-2,5(10)-estradien-17-on

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 28 - 35 g
- Housing: one animal per cage during study period
- 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

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 2, 10, 40 %
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 40 %. A preparation of a formulation >40 % 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.

Results and discussion

Positive control results:
The positive control, Alpha Hexyl Cinnamic Aldehyde, showed sensitizing potential. After treatment with it 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.
In addition, the positive control, Alpha Hexyl Cinnamic Aldehyde, showed irritating potential. The 'positive level' of ear swelling 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.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.78
Variability:
16.81
Test group / Remarks:
Exp. 5 (30% alpha hexyl cinnamic aldehyde)
Parameter:
other: weight index
Value:
1.7
Variability:
19.71
Test group / Remarks:
Exp. 5 (30% alpha hexyl cinnamic aldehyde)
Parameter:
SI
Value:
1.02
Variability:
34.64
Test group / Remarks:
Exp. 4 (40%)
Parameter:
SI
Value:
1.03
Variability:
27.54
Test group / Remarks:
Exp. 3 (10%)
Parameter:
SI
Value:
1.14
Variability:
38.66
Test group / Remarks:
Exp. 2 (2%)
Parameter:
SI
Value:
1
Variability:
18.86
Test group / Remarks:
Exp. 1 (0%)

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 (DMF) 1.00 ± 10.80 1.00 ± 18.86
2 % test item 0.95 ± 17.841.14 ± 38.66
10 % test item1.01 ± 25.44 1.03 ± 27.54
 40 % test item 1.03 ± 23.84 1.02 ± 34.64
30 % positive control 1.70* ± 19.71 1.78* ± 16.81

* 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 (DMF) 17.33 ± 3.7617.92 ± 2.87 1.00
2 % test item 17.17 ± 2.27 17.83 ± 4.021.00
10 % test item 17.50 ± 2.9818.00 ± 3.35 1.00
40 % test item 17.42 ± 2.9618.00 ± 5.30 1.00
30 % positive control 17.75 ± 4.25 23.92* ± 6.29 1.34

* statistically significant increase (p < 0.05)


 


Table 3: Ear weight


 



































 Groups Day 4 (ear weight in mg) (mean ± SD in %) Index Day 4
Vehicle (DMF) 13.26 ± 3.92 1.00
2 % test item 12.74 ± 6.63 0.96
10 % test item 13.23 ± 3.98 1.00
40 % test item 13.86 ± 5.99 1.05
30 % positive control 20.17 ± 8.911.58

* statistically significant increase (p < 0.05)


 


Only after treatment with the positive control, 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 (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).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance 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 40 % 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 substance.

Executive summary:

In a dermal sensitization study according to OECD TG 429 (24 April 2002) with D-ET-Dienon in Dimethylformamide, young adult female NMRI Mice (6/dose) were tested in the LLNA (OECD TG 429). Up to 40% of the test item was applied. Hexyl cinnamic aldehyde (CAS No 101-86-0) was used as positive control substance. Only after treatment with the positive control, 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.


 


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 substance.. In this study, the test item is not a dermal sensitizer.