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Administrative data

Description of key information

The test substance was found to be a skin sensitizer in the Local Lymph Node Assay.

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
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)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
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: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17.4-22.3g
- Housing: The animals were kept in groups in Makrolon Type II (pre-tests), and III (main study) cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and with granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10, 25%
No. of animals per dose:
5
Details on study design:
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.
- Administration of 3H-Methyl Thymidine: 3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytics, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.7 µCi of 3HTdR (equivalent to approximately 78.6 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Determination of Incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, 30827 Garbsen, Germany). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a -scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY®1, Schärfe System, Reutlingen, Germany). The values obtained were taken down manually.
- Determination of Ear Weights: After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Interpretation: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: First, that 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. Second, that the 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. Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response and the cutoff-value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cutoff-values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.
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).
The EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
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 test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003). Outliers were not identified.
However, both biological and statistical significance were considered together.
Positive control results:
EC3 = 20.1%
Parameter:
SI
Remarks on result:
other: Test substance concentration 5%: 1.16 Test substance concentration 10%: 1.49 Test substance concentration 25%: 6.83
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group: 314.2 DPM/animal Test substance concentration 5%: 365.8 DPM/animal Test substance concentration 10%: 466.8 DPM/animal Test substance concentration 25%: 2145.4 DPM/animal

- Viability / Mortality: No deaths occurred during the study period.

- 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 and biologically relevant increase in lymph node weight and –cell count was obtained in the high dose group in comparison to the vehicle control group (p<0.001). For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The index determined for the high dose group exceeded this threshold (index of 2.79).

- Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p=0.006). For BALB/c mice, a cutoff-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. Furthermore, the threshold value of 25% increase in ear weights for excessive local skin irritation mentioned in OECD guideline 429 was not exceeded in any group.

Lymph Node Weights

Test item concentration (%)

Lymph node weight (mg)

SD

Index

0

6.98

1.26

1.00

5

8.15

1.29

1.17

10

7.50

1.24

1.07

25

12.49*

1.50

1.79

Index = mean lymph node weight of test item group related to vehicle group

* statistically significant increase vs. control group (p < 0.001)

 

Lymph Node Cell Counts

Test item concentration (%)

Lymph node cell count (x10E6)

SD

Index

0

10.55

3.34

1.00

5

11.91

1.70

1.13

10

13.05

2.83

1.24

25

29.41*

8.01

2.79

Index = mean lymph node cell count of test item group related to vehicle group

* statistically significant increase vs. control group (p < 0.001)

 

Ear Weights

Test item concentration (%)

Ear weight (mg)

SD

Index

0

30.3

2.2

1.0

5

30.0

2.0

1.0

10

28.8

1.6

1.0

25

34.2*

2.7

1.1

Index = mean ear weight of test item group related to vehicle group

* statistically significant increase vs. control group (p < 0.001)

 

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a GLP compliant Local Lymph Node Assay, tested according to OECD guideline 429, 5 female CBA mice per dose were treated daily with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4 +1 v/v) by topical application to the dorsum of each ear lobe for three consecutive days. 25% was the highest concentration that could be applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments). A control group of five mice was treated with the vehicle only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio labeled thymidine (³H-methyl thymidine). Approximately five hours thereafter the mice were sacrificed and the draining auricular lymph nodes were excised and pooled per animal. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine. Stimulation Indices of 1.16, 1.49 and 6.83 were determined with the test item at concentrations of 5, 10, and 25%, respectively and a EC3 value of 14.2 (w/w) was calculated. Based on the described study and under the conditions reported, it is concluded that 2-ethyl-4-methylimidazoleis a sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the findings in the skin sensitisation study, the substance needs to be classified with Skin sens 1B, H317 according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.