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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
Bioassay Labor für biologische Analytik GmbH, Im Neuenheimer Feld 515/519, 69120 Heidelberg
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2 Dimethylimidazole
- BASF Test Item No.: 12/0074-1
- Batch Number: B 420
- Purity: 99.1 g/100 g
- Physical state, colour: Solid, melt / yellow
- Homogeneity: The test item was homogeneous by visual inspection.
- Storage conditions: Room temperature, avoid temperatures >35°C.
- Expiration Date: April 17, 2014

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: Pretest: 9 weeks, Main test: 10 weeks
- Weight at study initiation: Pre-test: 18.3 - 21.0 g; Main study: 19.1 - 24.2 g
- Housing: Single housing in Makrolon cage, type II, with bedding H 15005-29; Ssniff.Spezialitäten GmbH (Experimental AnimalDiets Inc., 59494 Soest, Germany)
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water: Tap water ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): Approx. 10
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-test: 100, 50 % w/w
Main test: 10, 25 and 50 % w/w
No. of animals per dose:
Pre-test: 2
Main test: 5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 442B. The highest test item concentration, which could be technically used, was 100%. The test item preparation was heated approx. 60 minutes at 60°C. The procedure was performed after consulting the sponsor. The preparation was administrated hand warm.
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in six animals (2 animals per test item concentration and vehicle, each). Six mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 100 and 50% (w/w) and with the vehicle acetone : olive oil (AOO, 4:1, v/v) once daily each on three consecutive days.

In the pre-test, the highest concentration that could be technically used was 100% (w/w). However both animals in the 100% group showed signs of local irritation as confirmed by ear weight measurement (>25%). The highest value of 159% exceeded the threshold value moderately. Both animals in the 100% group revealed an increase of 122% and 82% in ear thickness from day 0 to day 5 respectively. In addition the animals showed hardening of the ears, incrustations and scaling at the application side. Therefore this concentration could not be tested in the main study. At the test item concentration of 50% (w/w) both animals showed local findings like incrustations or scaling without exceeding the ear weight threshold. Signs of systemic toxicity were not observed in the pre-test. Therefore, the highest concentration tested in the main study was 50% (w/w) followed by 25% and 10% respectively.


MAIN STUDY
Test Item Preparation:
The test item preparations were produced on a weight per weight (w/w) basis. For better handling the test item preparations were heated approx. 60 minutes at 60°C and were dissolved in the vehicle AOO. The procedure was performed after consulting the sponsor. The preparations were administrated hand warm.
The positive control preparation was produced on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the positive control was soluble in the vehicle AOO.

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 10, 25, and 50% (w/w) in AOO. The application volume 25 μL/ear/day was spread over the entire dorsal surface (Ø~ 8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25 % α-hexyl cinnamaldehyde dissolved in acetone: olive oil (AOO 4:1 v/v).

Administration of BrdU:
BrdU was purchased from Roche Diagnostics GmbH. For injection it was dissolved in DPBS (10 mg/mL) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5 mg/per mouse/injection) were intraperitoneally injected.

Determination of Incorporated BrdU:
Approximately 24 hours after treatment with BrdU the mice were sacrificed and 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 a 70 μm nylon mesh. The lymph node cells were resuspended in 6 mL DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell suspensions of 100 000 cells / mL were adjusted.
The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit as follows (Roche Applied Science. Mannheim):
100 μL of the lymph node cell suspension (100 000 cells /mL) were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included inCell Proliferation Elisa Kit), anti BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution TMB (included inCell Proliferation Elisa Kit) was then added and allowed to produce chromogen. After stopping the substrate reaction the absorbance was measured at 450 nm with a reference wavelength of 690 nm (Absorbance-Reader SUNRISE, Tecan).

Determination of Lymph Node Weight and Cell Count:
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance (XS 204, Mettler Toledo, serial number 112 748 2217).
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (Casy Cell Counter, Innovatis). 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 (XS 204, Mettler Toledo, serial number 112 748 2217).

Interpretation of Raw Data:
The proliferative response of the lymph node cells is expressed as the mean SI. The SI is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group.

A test item is regarded as a sensitizer 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 BrdU at least 1.6-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. Furthermore, according to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation.

Observations:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.
Body weights: Pre-test and main experiment: prior to the first application and prior to sacrifice.
Ear thickness: In the pre-test prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).
Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical Analysis
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 BrdU values. For the calculations excel (Version 2010) was applied.
The EC1.6 value was calculated according to the equation: EC1.6 = (a-c) [(1.6-d)/(b-d)] + c
where EC1.6 is the estimated concentration of the test item required to produce a 1.6-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 1.6 on the local lymph node assay dose response plot.
A statistical analysis was conducted on the BrdU labeling, 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 Statistica (Version 10) was used. A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
As expected, a statistical and biological relevant increase in BrdU labelling, lymph node weight and lymph node cell count was determined in the positive control.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: Stimulation Index 1.0, 2.6, 4.8, and - (at concentrations of 0%, 10%, 25%, and 50 %, respectively)

Any other information on results incl. tables

Concentration (% w/w); Mean BrdU labeling Index:

0 %; 0.120

10 %; 0.314 (statistically significat increase vs. control group (p<0.05))

25 %; 0.579 (statistically significat increase vs. control group (p<0.05))

50 %; -

Viability / Mortality: A total of 3 animals died in the 50% dose group. One animal of the 50% group was found dead in the cage at day 2 of the experiment, and 2 other animals of the 50% group were found at day 5 of the experiment.

Clinical Signs: The animals did not show any relevant signs of systemic toxicity; however 3 animals died in the 50% group. Because the 50% dose group was excluded from the experiment, no further analysis was conducted in this group. In dose group 25% incrustations and scaling could be observed at day 5, scaling was also observed in dose group 10% at day 5.

Body Weights: The body weight of the animals recorded prior to the first application and prior to sacrifice were 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 increase in lymph node weights and - cell counts was observed in the 10% and 25% group in comparison to the vehicle control group.

Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights were not observed, whereupon in one animal of the 25% dose group the SI value of 1,23 (23%) reached nearly the cut-off value termed 1,25 (>25%), indicating slightly irritant properties.

According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria