L-arabinose

Endpoint summary

Description of key information

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) and does not activate keratinocytes (LuSens, OECD TG 422D). Therefore, the substance is not predicted to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 18 - October 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

yes

Remarks:

see "Any other information on Material and Methods"

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Dose Selection for Experiment I and II
In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 μM. For a test chemical which has no defined molecular weight, the final test item concentration 400 μg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). Since no cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment I and II:
269 μM, 323 μM, 388 μM, 465 μM, 558 μM, 670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM
In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction

Experimental Performance
Experiment I and II were performed in the same way. Experiment II serves only to confirm the results of experiment I. The exposure dates were 10. Oct. 2017 and 11. Oct. 2017. At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83000 (±10 %) cells/mL. 120 μL of the cell suspension were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h and 30 min in Experiment I and 24 h and 45 min in Experiment II. The treatment procedure was performed on both 96 well plates identically: After the incubation time the medium was removed from the cells and 150 μL medium no. 3 were added to each well. Afterwards 50 μL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution were added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 μL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5- Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability.
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 μL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 μL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometerFor calculation of the luciferase induction as well as the relative viability a validated Microsoft Excel® file was used.

Positive control results:

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values > 84 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.9 fold, negative control: 1.2 fold). However, the positive control induced a clear effect with an induction value of 4.5 fold in comparison to the solvent control.

Key result

Run / experiment:

other: Experiment I

Parameter:

other: fold Luciferase induction

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment II

Parameter:

other: fold Luciferase induction

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Acceptability of experiment I and II

Criteria	Found in Experiment I	Found in Experiment II
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.	Positive control Fold induction: 4.5 Relative viability: 84.7 %	Positive control Fold induction: 5.4 Relative viability: 94.2 %
The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.	Negative control: Fold induction: 1.2 Relative viability: 104.9 % Growth control: Fold induction: 0.9 Relative viability: 141.9 %	Negative control: Fold induction: 1.0 Relative viability: 112.0 % Growth control: Fold induction: 1.0 Relative viability: 136.5 %
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.	11.96 %	11.21 %
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.	12 concentrations are analysable	12 concentrations are analysable

Table 2: Summary of the Results of Experiment I

Parameter		Induction of Luciferase			Viability of the Cells
	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[μM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.12	11.96	100.0	5.69	5.69
Growth Control	-	0.9	0.07	7.80	141.9	6.20	4.37
Negative Control	5000	1.2	0.08	6.52	104.9	5.998	5.69
Positive Control	120	4.5	0.13	2.83	84.7	2.20	2.59
Test item	269	1.1	0.07	6.41	110.1	1.66	1.51
	323	1.0	0.08	8.19	104.9	3.07	2.92
	388	1.0	0.08	8.46	100.7	3.18	3.16
	465	1.0	0.04	4.00	101.3	2.14	2.11
	558	1.0	0.11	10.67	103.3	7.22	6.99
	670	1.0	0.03	3.32	101.4	6.13	6.05
	804	0.9	0.05	5.77	105.5	2.48	2.35
	965	1.0	0.05	5.62	94.0	1.14	1.21
	1157	0.9	0.06	6.58	93.4	3.24	3.47
	1389	0.9	0.05	5.86	98.1	0.65	0.66
	1667	0.9	0.06	7.53	95.8	2.08	2.17
	2000	0.9	0.06	6.88	100.2	1.20	1.20

Table 3: Summary of the Results of Experiment II

Parameter		Induction of Luciferase			Viability of the Cells
	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[μM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.11	11.21	100.0	5.89	5.89
Growth Control	-	1.0	0.08	8.22	136.5	4.39	3.22
Negative Control	5000	1.0	0.04	4.04	112.0	4.68	4.18
Positive Control	120	5.4	0.29	5.38	94.2	7.75	8.22
Test item	269	1.2	0.09	7.13	123.0	4.94	4.02
	323	1.1	0.05	4.03	111.3	3.21	2.88
	388	1.1	0.14	13.12	106.7	2.57	2.41
	465	1.1	0.09	8.52	103.8	2.77	2.67
	558	1.1	0.08	7.77	102.5	6.36	6.21
	670	1.0	0.02	2.52	102.3	4.68	4.58
	804	1.0	0.02	1.68	107.2	5.00	4.66
	965	1.0	0.06	6.63	110.7	3.14	2.84
	1157	1.1	0.06	5.27	106.4	1.34	1.26
	1389	1.0	0.07	7.02	102.8	0.88	0.86
	1667	1.0	0.17	17.22	99.8	7.77	7.78
	2000	0.9	0.03	3.46	99.9	2.58	2.58

Interpretation of results:

other: no activation of keratinocytes

Remarks:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In an in vitro skin sensitisation assay according to 442D, the test item did not induce luciferase activity in at least two independent experiment runs.

Executive summary:

In an in vitro skin sensitisation assay according to 442D, the potential of the test item to activate the Nrf2 transcription factor, by using the LuSens cell line was investigated. The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II:

269 μM, 323 μM, 388 μM, 465 μM, 558 μM, 670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM.

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration. EGDMA (120 μM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.D/L-lactic acid (5000 μM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control. The induction of the luciferase of the growth control was < 1.5 fold. Since all acceptability criteria of the assay were met the study is valid. No significant reduction of growth was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction. In all tested concentrations of the test item no substantial and reproducible dose dependent increase of luciferase induction was measured.

In conclusion, it can be stated that under the experimental conditions reported, the test did not have the potential to activate the Nrf2 transcription factor.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8th March - 14th March, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM (European Union Reference Laboratory for alternatives to animal test¬ing): "DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sen-sitation Testing."

Version / remarks:

29 June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Analytical instrument
HPLC system
Designation: HPLC_4
Components: Degasser G1322A, Quaternary pump G1311A, Autosampler G1313A, Column compartment G1316A, UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981
Usage and calibration following the corresponding SOP 114 00 526 in the current edition.

Column
An ACE Excel SuperC18 150x3 mm column with 3 um particles will be used. This column is used instead of the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline because it delivers substantially better peak shape for the peptides.

HPLC program
Eluent A: H2O + 0.1 % TFA
Eluent B: Acetonitrile + 0.085 % TFA
Gradient: See "Any other information on Materials and Methods".
Flow rate: 0.55 mL/min
Injection volume: 7 µL
Column temperature: 30 °C
Wavelength 1: 220 nm
Wavelength 2: 258 nm

Test System
Peptides with > 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion of positive controls. Additionally, a solvent control containing demineralised water instead of test item solution was prepared in triplicate (set C(water)) and also incubated and analysed together with the samples and was used for calculation of the depletion of the test item.

Positive control
Positive controls were treated identically as the test item. The following positive controls were used:
• Cinnamaldehyde (CAS 104-55-2, food grade >95 %) was used as 100 mM solution in acetonitrile for the cysteine peptide, Depletion range 60.8 - 100 %
• 2,3-Butanedione (CAS 431 -03-8, >97 %) was used as 100 mM solution in acetonitrile for the lysine peptide, Depletion range 10 - 45 %
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Dissolution of the test item
Two batches of 100 mM test item solutions were prepared: 45.3 mg test item were dissolved in 3.00 mL water (batch 20180312, used for Lys-peptide assay), 45.3 mg test item were dissolved in 3.00 mL acetonitrile (batch 20180308, used for Cys-peptide assay). The test item stock solution was freshly prepared for each assay.

Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
0.669 mM Cys-Peptide solution was prepared by dissolving 22.6 mg of the peptide in 45 mL phosphate buffer, pH 7.5. (batch 20180308)
0.667 mM Lys-Peptide solution is prepared by dissolving 23.3 mg of the peptide in 45.0 mL ammonium acetate buffer, pH 10.2. (batch 20180312)

Peptide calibration standards
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer (see chapter 7.2.1): 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM Peptide (Lys-Peptide) and 0.535 / 0.268 / 0.134 / 0.067 / 0.034 / 0.017 mM Pep-tide (Cys-Peptide). Calibration samples were analysed before the samples containing the test item. Blank dilution buffer was also measured.

Test item samples
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions. A final volume of 1 mL per sample was prepared for each sample.

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 23.6 h Lys-peptide and 22 h for Cys-peptide.
All three replicates of positive controls Cinnamaldehyde were turbid after incubation.

Calibration curve
From the peak areas of the peptide calibration standards detected at 220 nm, a linear cali¬bration curve was calculated using a Microsoft Excel® spreadsheet. The peptide concentration in all other samples was calculated using the linear regression (a = intercept, b = slope). Peptide concentration [mM] = (Peak area [mAU x min] –a) / b. As a peak purity criterion, the peak area ratio 220/258 nm should be constant over all analysed samples (100 ±10 %). For small peaks, this calculation may not be possible.

Peptide depletion
The peptide depletion was calculated for each individual sample using the following equation in a first step (equations are shown for Cys-Peptide, Lys-peptide is calculated analogously):

Peptide depletion c,i [%] = [1- (measured peptide peak area in sample / mean peptide peak area in solvent control C)] x 100

The mean peptide depletion of the Cys-peptide is calculated as follows:

Peptide depletion c = ∑c,I =1,2,3 Peptide depletion / 3

The mean peptide depletion of the test item is calculated using the following equation:

Mean peptide depletion [%] = (Peptide depletion c [%] + Peptide depletion k [%]) / 2

Key result

Run / experiment:

other: 1st run

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

2.56

Run / experiment:

other: 1st run

Parameter:

other: peptide depletion (%) cysteine peptide

Value:

1.06

Run / experiment:

other: 1st run

Parameter:

other: peptide depletion (%) lysine peptide

Value:

4.06

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: All acceptance criteria are fulfiled
The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.
The maximum standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item.
The maximum standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Table 1: Calculated peptide depletion values for the Lys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	24.28	26.01	2.69
Positive control Rep. 2	24.64
Positive control Rep. 3	29.11
Test item Rep. 1	6.11	4.06	1.89
Test item Rep. 2	2.40
Test item Rep. 3	3.66

Table 2: Calculated peptide depletion values for the Cys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	79.37	81.40	2.00
Positive control Rep. 2	81.47
Positive control Rep. 3	83.37
Test item Rep. 1	0.43	1.06	1.49
Test item Rep. 2	0.00
Test item Rep. 3	2.76

Interpretation of results:

other: no peptide binding

Conclusions:

The C-peptide depletion, caused by the test substance was determined to be 1.06%. The K-peptide depletion, caused by the test substance was determined to be 4.02%. The mean peptide depletion was 2.56%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows minimal l reactivity in the DPRA under the test conditions chosen.

Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item L-Arabinose towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in water was incubated 24 ± 2 h at 24.6 – 24.8 °C together with cysteine and lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel. The Cys-peptide depletion, caused by the test substance was determined to be 1.06%. The Lys-peptide depletion, caused by the test substance was determined to be 4.02%. The mean peptide depletion was 2.56%. Therefore, the DPRA predicition is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. 

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item L-Arabinose towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in water was incubated 24 ± 2 h at 24.6 – 24.8 °C together with cysteine and lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.The Cys-peptide depletion, caused by the test substance was determined to be 1.06%. The Lys-peptide depletion, caused by the test substance was determined to be 4.02%. The mean peptide depletion was 2.56%. Therefore, the DPRA predicition is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.

In a further in vitro skin sensitisation assay according to OECD 442D, the potential of the test item to activate the Nrf2 transcription factor, by using the LuSens cell line was investigated. The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II:

269 μM, 323 μM, 388 μM, 465 μM, 558 μM, 670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM.

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration. EGDMA (120 μM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control. DL-lactic acid (5000 μM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.The induction of the luciferase of the growth control was < 1.5 fold. Since all acceptability criteria of the assay were met the study is considered as valid. No significant reduction of growth was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction. In all tested concentrations of the test item no substantial and reproducible dose dependent increase of luciferase induction was measured.

In conclusion, it can be stated that under the experimental conditions reported, the test did not have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Conclusion:

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) and does not activate keratinocytes (LuSens, OECD TG 422D). Therefore, the substance is not predicted to be a skin sensitizer.

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 not classified as skin sensitising according to Regulation (EC) No 1272/2008 (CLP), as amended for the eleventh time in Commission Regulation (EU) 2018/669.