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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010-07-22
Deviations:
no
GLP compliance:
yes
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diethyl-1H-Imidazolium acetate (1:1)
EC Number:
692-759-5
Cas Number:
1040916-84-4
Molecular formula:
C7 H13 N2 . C2 H3 O2
IUPAC Name:
1,3-diethyl-1H-Imidazolium acetate (1:1)
Test material form:
liquid
Details on test material:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 4-5441-2379 VTA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no preparation needed

FORM AS APPLIED IN THE TEST:
undiluted test substance

PURITY
Purity: 97.4 ± 0.1 g/ 100 g

PH: ca. 6
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 4-5441-2379 VTA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no preparation needed

FORM AS APPLIED IN THE TEST:
undiluted test substance

PURITY
Purity: 97.4 ± 0.1 g/ 100 g

PH: ca. 6

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Aclimatization period at least 5 days before the first test substance application.

The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 30 – 70% for relative humidity. The day/night rhythm was 12 h light and 12 h darkness.
Type of cage: Makrolon cage, type II.
Feeding: Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
Drinking water: Tap water ad libitumTEST ANIMALS


- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.7 g – 20.8 g
- Housing: single housed
- Type of cage: Makrolon cage, type II.
- Diet: ad libitum, Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 2012-04-12 To: 2012-04-23

Study design: in vivo (LLNA)

Vehicle:
other: ethanol
Concentration:
Control group 1: 70% ethanol
Test group 2: test substance 10% in 70% ethanol (25 ul per ear)
Test group 3: test substance 25% in 70% ethanol (25 ul per ear)
Test group 4: test substance 50% in 70% ethanol (25 ul per ear)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 70% ethanol was used as the vehicle because good solubility of the preparation was achieved.

To determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Two mice per dose were treated with test substance concentrations of 25% and 50% or with the undiluted test substance on three consecutive days.

- Irritation:
Signs of local irritation were recorded on day 1, 2 and 5.
Indication of ear skin irritation was observed by ear measurements in the 50% concentration group.

- Systemic toxicity:
In the pre-test clinical signs were recorded after each application as well as on day 5.
The undiluted test substance caused mortality in both animals after the 2nd application.
Beside slightly reduced mean body weights in the animals of both concentrations concentrations, no signs of systemic toxicity were noticed during general observation.

- Ear measurement:
The ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally the weight of the pooled lymph nodes from both sides was determined for each animal.
The 50% concentration caused considerably increased ear weights and lymph node weights.
After application of the 25% test substance concentration the animals showed some increases in ear weights and lymph node weights
All in all, the following dose levels were selected for the main study: 10%, 25% and 50%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Murine Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
[SI: ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, termed the Stimulation Index (SI)]
1) The increase in 3H-thymidine incorporation by a factor of ≥ 3 (SI ≥ 3) and/or the increase in cell count by a factor of ≥ 1.5 (SI ≥ 1.5) as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
2) If a test substance shows a statistically significant and/or biologically relevant increase in 3H-thymidine incorporation, cell count, and/or lymph node weight as compared to the vehicle control in the presence of statistically significant and/or biologically relevant increased ear weights as indications of skin irritation, it is considered not to be a sensitizer.
3) If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in 3H-thymidine incorporation, cell count and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test substance may be considered to be a sensitizer or subjected to further investigations.
4) If statistically significant and/or biologically relevant increases in ear weights are accompanied by an increase in 3H-thymidine incorporation, cell count and/or lymph node weight, it cannot be ruled out that the lymph node response was caused by irritation and not by skin sensitization. Then, for identification of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control values (cf. section “Historical control data”) may be performed. If one or a combination of the measured parameters change statistical significance, evaluation on basis of the criteria described above may be possible. If the statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitization response.
5) If a test substance does not elicit a statistically significant increase in 3H-thymidine incorporation, cell count and/or lymph node weight but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
The test-substance preparation was produced on a weight per weight basis shortly before the application. After stirring with a magnetic stirrer the test substance was soluble in the vehicle.
Epicutaneous application (25 μL per ear, dorsal part of both ears, 3 consecutive applications (day 0 – day 2) to the same application site) is simulating dermal contact with the compound which is possible to occur under practical use conditions.
Positive control substance(s):
other: A concurrent positive control with a known sensitizer was not included in this study. Studies with alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in order to show that the test system is able to detect sensitizing compounds.
Statistics:
- stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements: ratio of the test group mean values for these parameters divided by those of the vehicle control group
- further statistical analyses: 3H-thymidine incorporation, cell count, lymph node weight and ear weight: WILCOXON - Test

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks:
³H-thymidine incorporation
Value:
1
Test group / Remarks:
1 - vehicle 70% ethanol
Parameter:
SI
Remarks:
³H-thymidine incorporation
Value:
1.2
Test group / Remarks:
2 - 10% in 70% ethanol
Parameter:
SI
Remarks:
³H-thymidine incorporation
Value:
4.13
Test group / Remarks:
3 - 25% in 70% ethanol
Parameter:
SI
Remarks:
³H-thymidine incorporation
Value:
4.61
Test group / Remarks:
4 - 50% in 70% ethanol
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
When applied as 25% and 50% preparations in 70% ethanol, the test substance induced a biologically relevant (increase above the cut off Stimulation Index of 3) and statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, the increase in the auricular lymph node cell counts was biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant at these concentrations.
In addition, a statistically significant increase in lymph node weights was noted at 10%

EC3 CALCULATION
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

CLINICAL OBSERVATIONS:
Compound residues were noted on the ear skin of the animals treated with the 50% concentration on day 5.

BODY/EAR WEIGHTS
The 10% and 25% test-substance preparations caused some and the 50% preparation a considerable increase in ear weights as indication of ear skin irritation. The increases were statistically significant at all concentrations.

The detailed result table is given in table 1.

Any other information on results incl. tables

Table 1: The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Test Group

Treatment

³H-thymidine

incorporation Stimulation Index

Cell Count

Stimulation Index

Lymph Node

Weight Stimulation Index

Ear Weight

Stimulation Index

1

vehicle 70% ethanol

1.00

 

1.00

 

1.00

 

1.00

 

2

10% in 70% ethanol

1.20

 

1.08

 

1.10

#

1.05

#

3

25% in 70% ethanol

4.13

##

1.70

##

1.63

##

1.19

##

4

50% in 70% ethanol

4.61

##

2.16

##

1.82

##

1.39

##

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The threshold concentration for sensitization induction was >10% < 25%.
The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 19.2% and 20.2%, respectively.

Because the lymph node response cannot be fully attributed to the ear skin irritation observed, it is concluded that EEIM Acetate exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.
Executive summary:

The skin sensitizing potential of test material was assessed 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/J mice each were treated with 10%, 25% and 50% w/w preparations of the test substance in 70% ethanol or with the vehicle alone. The high concentration was selected based on the presence of substance related mortality in a pre-test using the undiluted test substance.

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 for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone.

Three days after the last application the mice were injected into the tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-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 in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed during general observation.

When applied as 25% and 50% preparations in 70% ethanol, the test substance induced a biologically relevant (increase above the cut off Stimulation Index of 3) and statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, the increase in the auricular lymph node cell counts was biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant at these concentrations. There was a relevant and statistically significant increase in lymph node weights at these concentrations, as well. In addition, a statistically significant increase in lymph node weights was noted at 10%. The 10% and 25% test-substance preparations caused some and the 50% preparation a considerable increase in ear weights as indication of ear skin irritation. The increases were statistically significant at all concentrations. Compound residues were noted on the ear skin of the animals treated with the 50% concentration on day 5.

Because the lymph node response cannot be fully attributed to the ear skin irritation observed, it is concluded that the test material exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 19.2% and 20.2%, respectively.