Registration Dossier

Administrative data

Description of key information

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:
May - November 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:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V. Inc. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test: 11-12 weeks: Main study: 9-10 weeks
- Weight at study initiation: Pre-test: 19.7-20.1g; Main study: 18.4-22.9g
- Housing: grouped by test groups, Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: ad libitum 2018C Teklad Global 18 % protein rodent diet
- Water: ad libitum tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 (except for several hours)
- Humidity (%): 45-65 (except for several hours)
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
methyl ethyl ketone
Concentration:
10, 25, 50 %
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: experiment was performed according to the recommendations given by OECD 429
- Irritation: no excessive local skin irritation in 2 animals treated with 25 and 50 % on 3 consecutive days. From day 3 until 5, the animal treated with 50% test item concentration showed a very slight erythema of the ear skin (score 1). In addition, both animals showed substance residues during the course of the study. No signs of excessive local skin irritation or of systemic toxicity were observed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
a) exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at
least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index
b) data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either
local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Animals were treated topically on the dorsal surface of each ear (25 μL/ear/day) on 3 consecutive days
- 5 days after first application 78.2 μCi/mL 3HTdR were injected
- 5 h after injection animals were sacrificed
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell
count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical
program R Script DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with validated statistical program R Script Outlier.Rnw). No outlier was detected.
However, both biological and statistical significance were considered together.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
- Vehicle control: 1.0 - 10 %: 1.06 - 25 %: 1.59 - 50 %: 2.24* *Mean DPM value for the group was significantly higher than the corresponding control value. The p value for the analysis was 0.021. The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM per animal (2 lymph nodes) - Vehicle Control Group (MEK): 978.6 +/- 159.4 - 10% 2-Ethylimidazole: 1039.0 +/- 269.6 - 25% 2-Ethylimidazole: 1560.6 +/- 569.7 - 50% 2-Ethylimidazole: 2196.8 +/- 1098.0 Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals).

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were observed during the study period. On day 3 and 4, a slight erythema of the ear skin (score 1) was observed in the animals treated with 50% of the test item.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant but biologically not relevant increase in lymph node weights and –cell counts was observed in the highest dose group in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response (Ehling et al., 2005). The index determined for the lymph node cell count slightly exceeded this threshold in the high dose group (index of 1.61).

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice (Ulrich et al., 2001) was not reached or exceeded in any of the treated groups.

References:

Ehling G., Hecht, M., Heusener J., Gamer A.O., van Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R., Vohr H.-W. (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay 2nd round. Toxicology, 212, 69–79.

Ulrich P., Streich J., Suter W. (2001): Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals. Archives of Toxicology, 74, 733-744.

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The test item 2-Ethylimidazole was not a skin sensitiser under the test conditions of this study.
Executive summary:

In this study the test item 2-Ethylimidazole was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA, OECD 429) in mice. Test item solutions at different concentrations were prepared in the vehicle methyl ethyl ketone (MEK).

The LLNA is recommended as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a LLNA was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals showed neither signs of systemic toxicity nor mortality during the course of the study. On day 3 and 4, a slight erythema of the ear skin (score 1) was observed in the animals treated with 50% of the test item. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 1.06, 1.59 and 2.24 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in MEK, respectively. A clear dose response was observed.

Although a statistically significant increase in DPM value and also in lymph node cell count and lymph node weights was observed in the highest dose group in comparison to the vehicle control group, this was not considered to be biologically relevant as the S.I. determined for this concentration did not exceed the threshold value of 3. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was slightly exceeded in the high dose group (index of 1.61).

The test item 2-Ethylimidazole was thus not a skin sensitiser under the test conditions of this study.

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

In a GLP OECD429 study the test item 2-Ethylimidazole was assessed for its skin sensitising potential in the vehicle MEK at 10, 25 and 50 %.

A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, none of the indices exceeded the cut-off value of 1.1 for the ear weight index of BALB/C mice. In this study Stimulation Indices (S.I.) of 1.06, 1.59 and 2.24 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in MEK, respectively. A clear dose response was observed. Although a statistically significant increase in DPM value and also in lymph node cell count and lymph node weights was observed in the highest dose group in comparison to the vehicle control group, this was not considered to be biologically relevant as the S.I. determined for this concentration did not exceed the threshold value of 3. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was slightly exceeded in the high dose group (index of 1.61).

The test item 2-Ethylimidazole was thus not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result based on this LLNA the substance is not considered to be classified for sensitisation under Regulation (EC) No 1272/2008.