Registration Dossier

Administrative data

Description of key information

In a local lymphnode assay according to OECD guideline 429, the test item did not show skin sensitising properties.

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:
August 18 - October 10, 2016
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:
22th July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
Ola Hsd mice;
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: Young adult mice; 9-11 weeks
- Weight at study initiation: at starting: 17.7 – 23.0 g, The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging in small groups (acclimation period), 4 animals/cage(testing period); in Type II. Polypropylene / polycarbonate cages
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice, ssniff Spezialdiäten GmbH, 59494 Soest Germany, ad libitum.
- Water: tap water from watering bottles ad libitum.
- Acclimation period: 7 days
- Indication of any skin lesions: Only healthy animals (and not showing any sign of skin lesion) were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 from 6.00 a.m. to 6.00 p.m.

Vehicle:
dimethylformamide
Concentration:
0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v)
No. of animals per dose:
4 animals/ treatment- dose groups; 28 animals in the main test
Details on study design:
PRE-SCREEN TESTS:
Based on the preliminary test results the maximum attainable concentration (based on solubility) of 0.1 % (w/v) was used in the main test with the aim of testing the highest concentration possible. The test item was tested also at three additional, lower concentrations (0.05 %, 0.025 % and 0.01 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.
Animals in the treatment groups were treated with the negative (vehicle) controls (DMF or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.
In vivo Treatment
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles 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.

- Criteria used to consider a positive response:
A stimulation index of 3 or greater is an indication of a positive result (for calculation, see "Statistics").

Erythema Scores
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

TREATMENT PREPARATION AND ADMINISTRATION:
Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test: all animals treated were processed. Therefore no treatment group was excluded from the evaluation.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 L of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
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.

Preparation of Single Cell Suspension of Lymph Node Cells

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 re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR

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 loaded into the -scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The -counter expressed 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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 DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values.
Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated 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 = 13.8). 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.
Key result
Parameter:
SI
Value:
2.1
Variability:
p = 0.13, r = 0.87
Test group / Remarks:
at test item cocentration 0.1%
Key result
Parameter:
SI
Value:
1.1
Variability:
p = 0.13, r = 0.87
Test group / Remarks:
at test item cocentration 0.05%
Key result
Parameter:
SI
Value:
0.4
Variability:
p = 0.13, r = 0.87
Test group / Remarks:
at test item cocentration 0.025%
Key result
Parameter:
SI
Value:
1
Variability:
p = 0.13, r = 0.87
Test group / Remarks:
at test item cocentration 0.01%
Cellular proliferation data / Observations:
Since no failed treatment, sign of systemic toxicity or irritation was observed during the test no treatment group was excluded from the evaluation.
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were only observed in the positive control group. Appearance of the lymph nodes was normal in the negative control groups (AOO or DMF) and in the test item treated groups.
No significant lymphoproliferative response (SI ≥ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.1, 1.1, 0.4 and 1.0 at test item concentrations of 0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v), respectively.
Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.13, r = 0.87).

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score³ 3) or any other local effect were observed in any treatment group.

No significant, treatment related effect on body weights was observed during the test.

No significant lymphoproliferative response (SI ³ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations.

Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.13, r = 0.87).

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.

Table 2: Individual Ear Thickness Values and the Deviations from the Initial Values in the Dose Range Finding Test

Dose Group

Animal

Ears

Day 1*

Day 3$

Day 3

Day 6#

Day 6

 

Number

value (mm)

value (mm)

% deviation

value (mm)

% deviation

 

284

L

0.20

0.20

0.0

0.22

10.0

Leuco Sulfur Red 14

 

R

0.20

0.20

0.0

0.21

5.0

0.1 % in DMF

285

L

0.21

0.20

-4.8

0.20

-4.8

 

 

R

0.21

0.20

-4.8

0.20

-4.8

 

286

L

0.21

0.21

0.0

0.22

4.8

Leuco Sulfur Red 14

 

R

0.20

0.21

5.0

0.22

10.0

0.05 % in DMF

287

L

0.20

0.20

0.0

0.20

0.0

 

 

R

0.20

0.20

0.0

0.20

0.0

 

288

L

0.21

0.21

0.0

0.21

0.0

Leuco Sulfur Red 14

 

R

0.21

0.21

0.0

0.21

0.0

0.025 % in DMF

289

L

0.21

0.21

0.0

0.21

0.0

 

 

R

0.21

0.21

0.0

0.21

0.0

L = Left

R = Right

DMF =N,N-Dimethylformamide

 

* Ear thickness was measured prior to the first treatment.

$ Ear thickness was measured approximately 48 hours after the first treatment (prior to the third treatment).

# Ear thickness was measured at the end of the test.

 Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes in the Main Test

Table 3: Individual Body weight Development

Animal

Dose Group

Initial

Terminal

Body Weight

Number

 

Body Weight

Body Weight

Change

 

 

(g)

(g)

(%)

461

Vehicle control for the positive control:

19.8

20.2

2

473

AOO

21.0

22.1

5

474

 

18.6

18.5

-1

522

 

22.2

22.9

3

 

Mean

20.4

20.9

3

 

SD

1.5

2.0

 

462

Positive control:

19.5

20.8

7

475

25 % HCA

20.8

21.5

3

476

 in AOO

20.1

20.8

3

523

 

22.3

23.5

5

 

Mean

20.7

21.7

5

 

SD

1.2

1.3

 

463

Vehicle control for the test item:

19.3

20.2

5

477

DMF

21.1

21.7

3

524

 

23.0

22.7

-1

525

 

20.2

20.5

1

 

Mean

20.9

21.3

2

 

SD

1.6

1.2

 

464

Leuco Sulfur Red 14

22.6

21.8

-4

487

0.1 %

19.3

20.6

7

488

in DMF

21.2

23.0

8

533

 

20.4

21.2

4

 

Mean

20.9

21.7

4

 

SD

1.4

1.0

 

489

Leuco Sulfur Red 14

22.5

22.2

-1

490

0.05 %

21.0

22.5

7

491

in DMF

19.2

20.4

6

534

 

20.4

21.8

7

 

Mean

20.8

21.7

5

 

SD

1.4

0.9

 

492

Leuco Sulfur Red 14

20.9

21.6

3

493

0.025 %

17.7

17.3

-2

535

in DMF

21.7

23.2

7

536

 

20.1

20.9

4

 

Mean

20.1

20.8

3

 

SD

1.7

2.5

 

471

Leuco Sulfur Red 14

20.8

20.1

-3

494

0.01 %

22.2

23.8

7

495

in DMF

20.4

19.8

-3

537

 

18.0

18.8

4

 

Mean

20.4

20.6

1

 

SD

1.7

2.2

 

 

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMF =N,N-Dimethylformamide

 Table 4: Clinical Observations in the Main Test

Dose Group

Animal
Number

Days

1

2

3

4

5

6

PT

AT

PT

AT

PT

AT

Vehicle control for the positive control:
AOO

461

N

N

N

N

N

N

N

N

N

473

N

N

N

N

N

N

N

N

N

474

N

N

N

N

N

N

N

N

N

522

N

N

N

N

N

N

N

N

N

Positive control:

25 % HCA in AOO

462

N

N

N

N

N

N

N

N

N

475

N

N

N

N

N

N

N

N

N

476

N

N

N

N

N

N

N

N

N

523

N

N

N

N

N

N

N

N

N

Vehicle control for the test item:

DMF

463

N

N

N

N

N

N

N

N

N

477

N

N

N

N

N

N

N

N

N

524

N

N

N

N

N

N

N

N

N

525

N

N

N

N

N

N

N

N

N

Leuco Sulfur Red 14
0.1 % in DMF

464

N

N

N

N

N

N

N

N

N

487

N

N

N

N

N

N

N

N

N

488

N

N

N

N

N

N

N

N

N

533

N

N

N

N

N

N

N

N

N

Leuco Sulfur Red 14
0.05 % in DMF

489

N

N

N

N

N

N

N

N

N

490

N

N

N

N

N

N

N

N

N

491

N

N

N

N

N

N

N

N

N

534

N

N

N

N

N

N

N

N

N

Leuco Sulfur Red 14
0.025 % in DMF

492

N

N

N

N

N

N

N

N

N

493

N

N

N

N

N

N

N

N

N

535

N

N

N

N

N

N

N

N

N

536

N

N

N

N

N

N

N

N

N

Leuco Sulfur Red 14
0.01 % in DMF

471

N

N

N

N

N

N

N

N

N

494

N

N

N

N

N

N

N

N

N

495

N

N

N

N

N

N

N

N

N

537

N

N

N

N

N

N

N

N

N

PT = Prior to the treatment

AT = After the treatment

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 mixture (v/v)

DMF =N,N-Dimethylformamide

 

N = Normal (no symptoms observed)

 

Table 5: Erythema Scores in the Main Test

Dose Group

Animal Number

Ears

Days

1

2

3

4

5

6

PT

AT

PT

AT

PT

AT

Vehicle control for the positive control:
AOO

461

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

473

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

474

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

522

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Positive control:
25 % HCA in AOO

462

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

475

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

476

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

523

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Vehicle control for the test item:
DMF

463

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

477

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

524

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

525

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

L = Left                                                              R = Right

PT = Prior to treatment                                         AT = After the treatment

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMF =N,N-Dimethylformamide

HCA =a-Hexylcinnamaldehyde


Table 6:
Erythema Scores in the Main Test

Dose Group

Animal Number

Ears

Days

1

2

3

4

5

6

PT

AT

PT

AT

PT

AT

Leuco Sulfur Red 14
0.1 % in DMF

464

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

487

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

488

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

533

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Leuco Sulfur Red 14
0.05 % in DMF

489

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

490

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

491

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

534

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Leuco Sulfur Red 14
0.025 % in DMF

492

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

493

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

535

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

536

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Leuco Sulfur Red 14
0.01 % in DMF

471

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

494

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

495

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

537

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

L = Left                                                              R = Right

PT = Prior to treatment                                         AT = After the treatment

DMF =N,N-Dimethylfo

 Table 7: Visual Observations of the Lymph Nodes in the Main Test

Dose Group

Animal
Number

Appearance of Lymph Nodes

Vehicle control for the positive control:
AOO

461

N

473

N

474

N

522

N

Positive control:
25 % HCA in AOO

462

Larger than the relevant control (AOO)

475

Larger than the relevant control (AOO)

476

Larger than the relevant control (AOO)

523

Larger than the relevant control (AOO)

Vehicle control for the test item:
DMF

463

N

477

N

524

N

525

N

Leuco Sulfur Red 14
0.1 % in DMF

464

N

487

N

488

N

533

N

Leuco Sulfur Red 14
0.05 % in DMF

489

N

490

N

491

N

534

N

Leuco Sulfur Red 14
0.025 % in DMF

492

N

493

N

535

N

536

N

Leuco Sulfur Red 14
0.01 % in DMF

471

N

494

N

495

N

537

N

 

AOO = Acetone: Olive oil 4:1 (v/v) mixture

HCA =a-Hexylcinnamaldehyde

DMF =N,N-Dimethylformamide

N = Normal

Interpretation of results:
GHS criteria not met
Conclusions:
In a local lymphnode assay according to OECD guideline 429, the test item did not show skin sensitising properties.
Executive summary:

The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. The test item was applied to the skin of thedorsal surface of each earof young adult female CBA mice. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on the results the test item was formulated in N,N-Dimethylformamide (DMF) in the LLNA. The maximum achievable concentration (based on solubility) was 0.1 % (w/v) using ultrasonic dispersion and stirring. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 0.1 %, 0.05 %, 0.025 % or 0.01 % (w/v) formulations in DMF (4 animals /dose group). All formulations were adequately homogeneous during the application (apparently solutions, observed by the naked eye). Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed. The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 13.8) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.1, 1.1, 0.4 and 1.0 at test item concentrations of 0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.13, r = 0.87; evaluated by linear regression using SI values). According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 0.1 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item should not be considered as a skin sensitizer.

In conclusion, under the conditions of the present assay, the test item, tested at the maximum feasible concentration of 0.1 % (w/v, based on solubility) and also at concentrations of 0.05 %, 0.025 % or 0.01 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMF) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

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

The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. The test item was applied to the skin of the dorsal surface of each ear of young adult female CBA mice. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on the results the test item was formulated in N,N-Dimethylformamide (DMF) in the LLNA. The maximum achievable concentration (based on solubility) was 0.1 % (w/v) using ultrasonic dispersion and stirring. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 0.1 %, 0.05 %, 0.025 % or 0.01 % (w/v) formulations in DMF (4 animals /dose group). All formulations were adequately homogeneous during the application (apparently solutions, observed by the naked eye). Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed. The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 13.8) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.1, 1.1, 0.4 and 1.0 at test item concentrations of 0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.13, r = 0.87; evaluated by linear regression using SI values). According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 0.1 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item should not be considered as a skin sensitizer.In conclusion, under the conditions of the present assay, the test item, tested at the maximum feasible concentration of 0.1 % (w/v, based on solubility) and also at concentrations of 0.05 %, 0.025 % or 0.01 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMF) was shown to have no skin sensitization potential in the Local Lymph Node Assay

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is considered not to be classified for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.