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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:
From September 4th to September 11th, 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol
EC Number:
246-042-5
EC Name:
α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol
Cas Number:
24155-42-8
Molecular formula:
C11H10Cl2N2O
IUPAC Name:
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: good conventional hygienic level. No further data.
- Age at study initiation: 11-12 weeks old (at start of the preliminary test and at the start of the main test)
- Weight at study initiation: 17.0-22.3 g. The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging in small groups (4 animals/cage), in Type II. Polypropylene / polycarbonate cages, with deep wood sawdust bedding, to allow digging and other normal rodent activities. Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg (Germany) Holzmühle 1) was available to animals during the study.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water (e.g. ad libitum): tap water from municipal supply, ad libitum.
- Acclimation period: 7 days.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
Source: Scharlau, Batch: 12673903.
Concentration:
25 %, 10 %, 5 % and 2.5 % (w/v).
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Solubility of the test item was evaluated in the following vehicles: Acetone: Olive oil 4:1 mixture (v/v) (AOO), N,N-Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), Methyl ethyl ketone (MEK), Absolute ethanol: water 7:3 mixture (EtOH). The best solubility was achieved in DMF with a maximum concentration of 25 % (w/v); based on concentration series recommended by the guidelines.
- Irritation: The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used. The test item was formulated in N,N-Dimethylformamide (DMF) and evaluated at concentrations of 25 % and 10 % (w/v). No signs of significant irritation (indicated by an erythema score≥ 3 and/or an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the treatment groups.
- Systemic toxicity: No mortality or any signs of systemic toxicity were observed during the preliminary test. No significant, treatment related effect on body weights was observed.
- Ear thickness measurements: Measurement of ear thickness was taken using digital micrometer on Day 1 (predose), Day 3 (approximately 48 hours after the first dose) and Day 6. See Tables in 'any other information of materials and methods incl. tables'.
- Erythema scores: See Tables in 'any other information of materials and methods incl. tables'.
Since no systemic toxicity or sign of irritation was observed during the preliminary test, the 25 % concentration was selected to be used as the maximum in the main test.

MAIN STUDY
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item (in DMF), the positive control substance (positive control group, in AOO) or the vehicles (DMF or AOO as negative control groups) using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate with purified water) containing 20 µCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes). Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Clinical observations: During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.
- Body weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result. The strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION: A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell
strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and resuspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement. For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA. DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 8.1). The results
of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.3
Test group / Remarks:
25% test item
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10% test item
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
5% test item
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
2.5% test item
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: No significant (SI ≥ 3) lymphoproliferative response was noted for the test item in DMF at the tested concentrations. The observed stimulation index values were 2.3, 1.6, 2.0 and 1.2 at test item concentrations of 25 %, 10 %, 5 % and 2.5 %, respectively. See 'Any other information on results'.

DETAILS ON STIMULATION INDEX CALCULATION: The SI = the DPN (disintegration per minute divided by number of lymph nodes) of a treated (positive control or test item) group divided by the DPN of the respective negative control group.

EC3 CALCULATION: Based on the results, the EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated. Dose-response relationship was evaluated by linear regression. All calculations were made by Microsoft Excel Software.

CLINICAL OBSERVATIONS: No significant, treatment related effect on body weights was observed during the test. Visually larger than the control lymph nodes were observed in the positive control group only. Appearance of the lymph nodes was normal in both negative control groups (DMF and AOO) and in the test item treated groups.

BODY WEIGHTS: See tables in 'Any other information on results'.

Any other information on results incl. tables

Table 5. DPM, DPN and SI values for all groups in the Main Test.

Dose

Group

DPM/group

Group* DPM

DPN (DPM/Node)

SI

Vehicle control (AOO)

17750

17710.5

2213.8

1.0

Positive control

(25% HCA in AOO)

144132

144092.5

18011.6

8.1

Vehicle control (DMF)

7226

7186.5

898.3

1.0

Test item 25% in DMF

16576

16536.5

2067.1

2.3

Test item 10% in DMF

11831

11791.5

1473.9

1.6

Test item 5% in DMF

14333

14293.5

1786.7

2.0

Test item 2.5% in DMF

8751

8711.5

1088.9

1.2

HCA = α-Hexylcinnamaldehyde; AOO = Acetone:Olive oile 4:1 (v/v) mixture; DMF = N, N-Dimethylformamide.

 

Table 6. Individual Body Weights of the Animals with Group Means in the Main Test.

Animal No.

Test Group Name

Initial Body Weight

(g)

Terminal Body Weight

(g)

Body weight change

(%)

235

Vehicle control for the positive control: AOO

17.8

19.3

8

236

20.5

21.0

2

264

19.5

19.5

0

265

21.7

22.1

2

Mean

19.9

20.5

3

237

Positive control:

25% HCA in AOO

21.2

21.9

3

266

17.0

17.2

1

267

20.5

20.4

0

268

19.6

19.8

1

Mean

19.6

19.8

1

244

Vehicle control for the test item: DMF

20.3

20.3

0

245

21.2

21.4

1

269

19.1

19.7

3

270

18.5

19.1

3

Mean

19.8

20.1

2

246

Test item 25% in DMF

20.4

21.0

3

247

21.2

20.8

-2

281

19.2

20.1

5

282

18.6

19.3

4

Mean

19.9

20.3

2

248

Test item 10% in DMF

18.8

18.5

-2

283

22.3

21.6

-3

284

20.3

20.4

0

285

19.1

20.1

5

Mean

20.1

20.2

0

249

Test item 5% in DMF

19.8

19.4

-2

250

21.2

21.3

0

286

17.2

17.7

3

287

20.5

19.9

-3

Mean

19.7

19.6

-1

251

Test item 2.5% in DMF

19.1

18.9

-1

288

21.3

22.0

3

289

20.6

21.3

3

290

18.3

19.0

4

Mean

19.8

20.3

2

HCA = α-Hexylcinnamaldehyde; AOO = Acetone:Olive oile 4:1 (v/v) mixture; DMF = N, N-Dimethylformamide.

 

Table 7. Clinical Observations in the Main Test.

Animal No.

Test Group Name

Day 1

(1stT)

Day 2

(2nd T)

Day 3

(3rd T)

Day 4

Day 5

Day 6

PT

AT

PT

AT

PT

AT

235

Vehicle control

for the

positive control:

AOO

N

N

N

N

N

N

N

N

N

236

N

N

N

N

N

N

N

N

N

264

N

N

N

N

N

N

N

N

N

265

N

N

N

N

N

N

N

N

N

237

Positive control:

25% HCA in AOO

N

N

N

N

N

N

N

N

N

266

N

N

N

N

N

N

N

N

N

267

N

N

N

N

N

N

N

N

N

268

N

N

N

N

N

N

N

N

N

244

Vehicle control

for the test item:

DMF

N

N

N

N

N

N

N

N

N

245

N

N

N

N

N

N

N

N

N

269

N

N

N

N

N

N

N

N

N

270

N

N

N

N

N

N

N

N

N

246

Test item 25%

in DMF

N

N

N

N

N

N

N

N

N

247

N

N

N

N

N

N

N

N

N

281

N

N

N

N

N

N

N

N

N

282

N

N

N

N

N

N

N

N

N

248

Test item 10%

in DMF

N

N

N

N

N

N

N

N

N

283

N

N

N

N

N

N

N

N

N

284

N

N

N

N

N

N

N

N

N

285

N

N

N

N

N

N

N

N

N

249

Test item 5%

in DMF

N

N

N

N

N

N

N

N

N

250

N

N

N

N

N

N

N

N

N

286

N

N

N

N

N

N

N

N

N

287

N

N

N

N

N

N

N

N

N

251

Test item 2.5%

in DMF

N

N

N

N

N

N

N

N

N

288

N

N

N

N

N

N

N

N

N

289

N

N

N

N

N

N

N

N

N

290

N

N

N

N

N

N

N

N

N

HCA = α-Hexylcinnamaldehyde; AOO = Acetone:Olive oile 4:1 (v/v) mixture; DMF = N, N-Dimethylformamide.

T = treatment; PT: prior to treatment; AT: after treatment; N: normal (no signs of toxicity observed).

 

Table 8. Erythema Scores in the Main Test.

Animal No.

Test Group Name

Days

Ears

1

2

3

4

5

6

PT

AT

PT

AT

PT

AT

235

Vehicle control

for the

positive control:

AOO

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

236

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

264

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

265

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

237

Positive control:

25% HCA in AOO

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

266

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

267

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

268

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

244

Vehicle control

for the test item:

DMF

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

245

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

269

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

270

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

246

Test item 25%

in DMF

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

247

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

281

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

282

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

248

Test item 10%

in DMF

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

283

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

284

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

285

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

249

Test item 5%

in DMF

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

250

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

286

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

287

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

251

Test item 2.5%

in DMF

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

288

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

289

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

290

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

HCA = α-Hexylcinnamaldehyde; AOO = Acetone:Olive oile 4:1 (v/v) mixture; DMF = N, N-Dimethylformamide.

L: left; R: right; PT: prior to treatment; AT: after treatment.

Table 9. Visual Observations of the Lymph Nodes in the Main Test.

Animal No.

Test Group Name

Appearance of lymph nodes

235

Vehicle control

for the

positive control:

AOO

N

 

236

N

 

264

N

 

265

N

 

237

Positive control:

25% HCA in AOO

Larger than the control

 

266

Larger than the control

 

267

Larger than the control

 

268

Larger than the control

 

244

Vehicle control

for the test item:

DMF

N

 

245

N

 

269

N

 

270

N

 

246

Test item 25%

in DMF

N

 

247

N

 

281

N

 

282

N

 

248

Test item 10%

in DMF

N

 

283

N

 

284

N

 

285

N

 

249

Test item 5%

in DMF

N

 

250

N

 

286

N

 

287

N

 

251

Test item 2.5%

in DMF

N

 

288

N

 

289

N

 

290

N

 

HCA = α-Hexylcinnamaldehyde; AOO = Acetone:Olive oile 4:1 (v/v) mixture; DMF = N, N-Dimethylformamide.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria.
Conclusions:
Based on the test results (SI = 2.3 at the maximum attainable dose), the test item is not sensitising.
Executive summary:

The skin sensitisation potential of the test item was studied according to OECD 429 / EU Method B.42, under GLP conditions. The test item was tested at the solubility limit of 25 % and at three lower concentrations (10 %, 5 % and 2.5 %) in N,N-Dimethylformamide (DMF). Appropriate positive (25% α-Hexylcinnamaldehyde in AOO) and negative controls (DMF, AOO) were employed. Four female CBA/Ca mice were tested in each group. Each mouse was topically treated with 25 µL of the appropriate formulations using a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or significant, treatment related effects on body weights or any other sign of systemic toxicity were observed in any treatment group during the test. No signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group. The stimulation index values were 2.3, 1.6, 2.0 and 1.2 at test item concentrations of 25 %, 10 %, 5 % and 2.5 %, respectively. The lack of a significantly increased proliferation up to the maximum attainable concentration of 25 % (w/v) and the lack of a significant, dose-related response are considered evidence that the test item is not sensitising. Therefore, the test item is not sensitising.