p-menth-1-en-4-ol

Endpoint summary

Description of key information

LuSens (2018): activation of keratinocytes: positive

DPRA (2018): protein reactivity: positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar 2018 - Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Feb 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) 2017/735 B.59 In Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA)

Version / remarks:

Feb 2017

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
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identity: Confirmed
- Purity: 97.3%
- Physical state / color: liquid / colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, under light exclusion
- Stability under test conditions: The stability under storage conditions was guaranteed
- Homogeneity: The test substance was homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test substance preparations were prepared on a weight per volume basis. The test substance was prepared at a 100 mM concentration in acetonitrile
- Reason for vehicle: The test substance was soluble in acetonitrile.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM:
Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

CONTROLS:
- Negative control (NC): vehicle control = acetonitrile
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA), prepared as a 50 mM solution in acetonitrile.
- Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide.

EXPERIMENTAL PROCEDURE:
- Number of replicates: 3
- Analytical method: HPLC with gradient elution and UV-detection at 220 nm

DATA EVALUATION:
- Calculations: performed with EXCEL
- Peptide depletions peak areas at 220 nm were used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity.

ACCEPTANCE CRITERIA:
- The standard calibration curve should have an r² > 0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- In addition the positive control should cause depletion of both peptides comparable to historic data.

Run / experiment:

other: 1

Parameter:

other: peptide depletion [%]

Value:

95.27

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: peptide depletion [%]

Value:

95.26

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: peptide depletion [%]

Value:

95.35

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Peak areas, peptide concentration and peptide depeltion of NC, PC and the test substance for cysteine-peptide

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean SD
NC: ACN	-0.63	0.26	0.37	0.00 0.55
Terpinen-4-ol	> 95.27	> 95.26	> 95.35	> 95.29 0.05
PC: EGDMA in ACN	63.56	64.83	67.39	65.26 1.95

Mean peptide depletion is not applicable for the test substance due to co-elution.

The mean area ratio 220 nm/ 258 nm of the 9 vehicle control samples of sets B and C was calculated to be 30.4. Hence, the area ratio 220 nm/ 258 nm of the test-substance samples correspond to 5.6% to 5.9% of the mean of the vehicle controls, demonstrating interference.

Calculation of depletion values was not senseful due to the co-elution of the test substance with the lysine peptide and as the peak area values at 220 nm are in the margin of the NC.

Co-elution of the test substance and both peptides occurred as demonstrated by the values of the area ratios 220 nm/258 nm. Evaluation of the cysteine-peptide reaction was possible despite the co-elution as very small peak areas were detected at the retention time of the cysteine. For the lysine-peptide reaction evaluation was not possible, as the peak areas detected at 220 nm are in the margin of the NC and it is not possible to separate a possible peptide peak from a test-substance peak.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the observed results and applying the cysteine 1:10 prediction model it was concluded that the test substance shows moderate to high chemical reactivity in the DPRA under the test conditions chosen.
Based on the observed results and applying the evaluation criteria, the test substance is peptide reactive and activates keratinocytes. Thus, the test substance is predicted to be a skin sensitizer.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

However, in the current case for the test substance the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

DPRA: The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides.

Further, in order to detect possible interference of the test substance with the peptides, a coelution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm. The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

Co-elution of test substance and both peptides was present. However, due to the high peptidedepletion value obtained for the C-peptide, an evaluation of the C-peptide reaction is possible. The mean C-peptide depletion, caused by the test substance was determined to be >95.29%. The mean K-peptide depletion, caused by the test substance was not evaluable due to coelution of the test substance with the peptide (interference). Due to the interference observed in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 2018 - Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Feb 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) 2017/735 of Feb 2017

Version / remarks:

Feb 2017

Deviations:

no

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
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 01767-1010
- Identity: Confirmed
- Purity: 97.3%
- Physical state / color: liquid / colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, under light exclusion
- Stability under test conditions: The stability under storage conditions was guaranteed
- Homogeneity: The test substance was homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test substance preparations were prepared on a weight per volume basis within 4 hours of application. The test substance was topped up with 4% DMSO in culture medium. Test substance preparations were prepared by stirring.
- Reason for vehicle: The test substance was soluble in 4% DMSO in culture medium 3.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

SELECTION OF CONCENTRATIONS IN A PRE-TEST:
- Concentrations: 0.5 - 2000 µM
- Cytotoxicity test: MTT
- CV75 value (= estimated concentration that affords 75% cell viability): determined by linear regression to be 451 µM

SELECTION OF CONCENTRATIONS FOR MAIN STUDY:
- Highest concentration: 1.2³-fold of CV75
- Additional concentrations: 1:1.2 serial dilution of the highest concentration

FURTHER CHOICE OF CONCENTRATIONS
As no cytotoxicity below 70% was observed in all test-substance concentrations of the 1st experiment and as no luciferase activity above 1.5-fold induction of statistical significance with respect to the vehicle control was observed in two consecutive concentrations, the selected concentrations were too low. Hence, higher concentrations were tested in further experiments.

TEST SYSTEM:
- Cell line: LuSens (Human transgenic keratinocyte cell line derived from HaCaT cells)

CONTROLS:
- Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
- Vehicle control (VC): 1% DMSO in culture medium 3
- Blank control: culture medium 3 without cells
- Basal control: culture medium 3 with cells

EXPERIMENTAL PROCEDURE:
- Exposur eperiod: 48 +/- 1 hour
- Luciferase assay: Luminescence was measured in the luminometer
- Cytotoxicity: MTT

DATA EVALUATION:
- Relative survival rate od the CV75-value: linear regression

ACCEPTANCE CRITERIA:
- A tested concentration is not further evaluated when relative viability is less than 70%.
- The cell viability of VC cells must yield at least 85%.
- The mean of the positive control EGDMA should achieve ≥ 2.50 fold-induction and the mean of the LA < 1.50 and the mean of the viability must be ≥70%.
- The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
- The mean of the basal expression of the cells must be <1.50 fold-induction as compared to the solvent control.
- In addition, positive, negative and vehicle control data should lie within the range of the historic data.

Run / experiment:

other: Experiment 2

Parameter:

other: fold induction

Value:

1.52

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: fold induction

Value:

1.57

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: fold induction

Value:

1.58

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: fold induction

Value:

1.67

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: fold induction

Value:

1.72

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experimet 5, plate 1

Parameter:

other: fold induction

Value:

1.66

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 5, plate 1

Parameter:

other: fold induction

Value:

1.75

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 5, plate 1

Parameter:

other: fold induction

Value:

2.25

Vehicle controls validity:

valid

Remarks:

vehicle control is negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Table 1: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 1)

Concentration (test substance) [µM]	foldindu	ction	1^stexperiment rel. viability [%]			t-test
	mean	SD	mean	SD	p-value		markers
218	0.89	0.01	101	3	0.000		**
261	1.16	0.03	92	2	0.000		**
313	1.21	0.08	88	6	0.017		*
376	1.09	0.07	78	2	0.069		n.s.
451	1.13	0.09	80	3	0.051		n.s.
542	1.43	0.06	80	2	0.001		**
650	1.33	0.08	70	3	0.004		**
780	1.46	0.10	71	0	0.004		**
VC	1.00	0.09	100	4	-		-
EGDMA 90.8 µM	4.71	0.67	95	3	0.000		**
LA 5000 µM	0.99	0.07	105	4	0.416		n.s.

Table 2: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 2)

Concentration(testsubstance) [µM]	foldindu	ction	2^ndexperiment rel. viability [%]			t-test
	mean	SD	mean	SD	p-value		markers
376	1.21	0.06	86	1	0.003		**
451	1.41	0.06	79	3	0.000		**
542	1.52	0.20	83	4	0.022		*
650	1.37	0.12	77	5	0.013		*
780	1.57	0.06	72	2	0.000		**
936	1.51	0.06	65	1	0.000		**
1123	1.68	0.10	55	1	0.001		**
1348	2.23	0.11	44	1	0.001		**
VC	1.00	0.09	100	5	-		-
EGDMA 90.8 µM	6.51	0.77	81	3	0.000		**
LA 5000 µM	0.93	0.06	97	3	0.038		*

Table 3: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 3)

Concentration (test substance) [µM]	fold indu	ction	3^rdexperiment rel. viability [%]			t-test
	mean	SD	mean	SD	p-value		markers
376	1.43	0.14	83	4	0.014		*
451	1.29	0.07	76	2	0.003		**
542	1.58	0.31	81	3	0.041		*
650	1.67	0.10	78	4	0.002		**
780	1.72	0.15	72	2	0.006		**
936	2.21	0.34	68	2	0.012		*
1123	2.27	0.28	59	2	0.007		**
1348	2.68	0.11	51	1	0.000		**
VC	1.00	0.08	100	4	-		-
EGDMA 90.8 µM	4.81	0.33	86	5	0.000		**
LA 5000 µM	0.91	0.03	98	4	0.001		**

The EC1.50 was calculated by linear regression from the results of the 451 μM and the 542 μM concentrations to be 516 μM.

The 4th experiment, conducted with concentrations of 376 μM up to 1348 μM was not evaluable, as only one test-substanc concentration had a relative viability above 70%

Table 4: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 5, plate 1)

Concentration(testsubstance) [µM]	foldindu	ction	5^thexperiment plate 1 rel. viability [%]			t-test
	mean	SD	mean	SD	p-value		markers
376	1.43	0.11	85	4	0.007		**
451	1.39	0.04	83	4	0.000		**
542	1.66	0.10	79	3	0.002		**
650	1.75	0.08	78	2	0.001		**
780	2.25	0.18	76	3	0.003		**
936	2.57	0.31	69	1	0.006		**
1123	3.69	0.03	61	2	0.000		**
1348	4.81	0.84	45	3	0.008		**
VC	1.00	0.08	100	5	-		-
EGDMA 90.8 µM	4.99	0.38	87	5	0.000		**
LA 5000 µM	0.85	0.09	102	2	0.008		**

Table 5: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 5, plate 2)

Concentration(testsubstance) [µM]	foldindu	ction	5^thexperiment plate 2 rel. viability [%]			t-test
	mean	SD	mean	SD	p-value		markers
87	1.14	0.02	98	5	0.000		**
105	1.19	0.02	94	1	0.000		**
126	1.25	0.05	90	3	0.000		**
151	1.19	0.11	97	5	0.041		*
181	1.21	0.14	93	4	0.053		n.s.
218	1.25	0.12	86	3	0.024		*
261	1.20	0.10	89	4	0.024		*
313	1.17	0.06	87	1	0.004		**
VC	1.00	0.11	100	4	-		-
EGDMA 90.8 µM	4.76	0.58	91	4	0.000		**
LA 5000 µM	0.93	0.09	102	6	0.098		n.s.

The EC1.50 was calculated by linear regression from the results of the 451 μM and the 542 μM concentrations to be 488 μM.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this,
it has to be concluded that the test substance has a keratinocyte activating potential. Based on the observed results and applying the evaluation criteria, the test substance is peptide reactive and activates keratinocytes. Thus, the test substance is predicted to be a skin sensitizer.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

LuSens: The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca.48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP)

was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 451 μM (corresponding to test substance as provided by the sponsor).

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid and evaluable experiments were performed. Two further experiments were valid but not useful for evaluation. They are included in the results section but not in the summary table. The following results were observed:

The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

Relative luciferase fold induction and concurrent relative viability determined in the main experiments are summarized in table 1.

Concentration (test substance) [µM]	fold induction	2^ndexperiment rel. viability [%		t-test
	mean	mean	p-value		markers
376	1.21	86	0.003		**
451	1.41	79	0.000		**
542	1.52	83	0.022		*
650	1.37	77	0.013		*
780	1.57	72	0.000		**
936	1.51	65	0.000		**
1123	1.68	55	0.001		**
1348	2.23	44	0.001		**
VC	1.00	100	-		-
EGDMA 90.8 µM	6.51	81	0.000		**
LA 5000 µM	0.93	97	0.038		*

Concentration(test substance) [µM]	fold induction	3^rdexperiment rel. viability [%		t-test
	mean	mean	p-value		markers
376	1.43	83	0.014		*
451	1.29	76	0.003		**
542	1.58	81	0.041		*
650	1.67	78	0.002		**
780	1.72	72	0.006		**
936	2.21	68	0.012		*
1123	2.27	59	0.007		**
1348	2.68	51	0.000		**
VC	1.00	100	-		-
EGDMA 90.8 µM	4.81	86	0.000		**
LA 5000 µM	0.91	98	0.001		**

Concentration(test substance) [µM]	fold induction	5^thexperiment plate 1 rel. viability [%		t-test		Concentration (test substance) [µM]	fold induction	5^thexperiment plate 2 rel. viability [%		t-test
	mean	mean	p-value		markers		mean	mean	p-value		markers
376	1.43	85	0.007		**	87	1.14	98	0.000		**
451	1.39	83	0.000		**	105	1.19	94	0.000		**
542	1.66	79	0.002		**	126	1.25	90	0.000		**
650	1.75	78	0.001		**	151	1.19	97	0.041		*
780	2.25	76	0.003		**	181	1.21	93	0.053		n.s.
936	2.57	69	0.006		**	218	1.25	86	0.024		*
1123	3.69	61	0.000		**	261	1.20	89	0.024		*
1348	4.81	45	0.008		**	313	1.17	87	0.004		**
VC	1.00	100	-		-	VC	1.00	100	-		-
EGDMA 90.8 µM	4.99	87	0.000		**	EGDMA 90.8 µM	4.76	91	0.000		**
LA 5000 µM	0.85	102	0.008		**	LA 5000 µM	0.93	102	0.098		n.s.

A total of 3 valid and evaluable experiments were performed. Two further experiments were valid but not useful for evaluation and thus not shown in the summary table. Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 516 μM (experiment 3) and 488 μM (experiment 5), respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of the test substance was assessed using a combination of the following three in vitro methods, addressing the key events of the adverse outcome pathway (AOP) for skin sensitization as defined by the OECD (OECD, 2012):

• protein reactivity (DPRA)


• activation of keratinocytes (LuSens) and


• activation of dentritic cells (h-CLAT)

In the current case for the test substance the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

Protein reactivity of the test substance was determined in a Direct Peptide Reactivity Assay (DPRA) according to OECD TG 442C, complying to GLP principles (2018). The mean C-peptide depletion, caused by the test substance was determined to be >95.29%. The mean K-peptide depletion, caused by the test substance was not evaluable due to coelution of the test substance with the peptide (interference). Due to the interference observed in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

Based on the observed results and applying the evaluation criteria, the test substance is peptide reactive and activates keratinocytes. Thus, the test substance is predicted to be a skin sensitizer

In a second study, the activation of keratinocytes due to treatment with the test substance was investigated in an ARE-Nrf2 Luciferase Test (LuSens) according to OECD TG 442D, in compliance with GLP (2018). A total of 3 valid and evaluable experiments were performed. Two further experiments were valid but not useful for evaluation and thus not shown in the summary table. Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 516 μM (experiment 3) and 488 μM (experiment 5), respectively.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this, it has to be concluded that the test substance has a keratinocyte activating potential. Based on the observed results and applying the evaluation criteria, the test substance is peptide reactive and activates keratinocytes. Thus, the test substance is predicted to be a skin sensitizer.

Justification for classification or non-classification

The available data on skin sensitisation of the test substance warrant a classification as skin sensitizer (Skin Sens Cat. 1) according to Regulation (EC) 1272/2008.