Levomenol

Endpoint summary

Description of key information

Different in vitro and in vivo studies on skin sensitisation are available for (-)-α-Bisabolol (CAS 23089 -26 -1, levomenol) and the synthetic racemic (+/-)-α-Bisabolol.

(+/-)-α-Bisabolol (source substance) is considered to be a suitable read across substance for (-)-α-Bisabolol (target substance).

In vitro, conflicting results were obtained with (-)-α-Bisabolol: While the KeratinoSens (Ref. 7.4.1 -4) was negative, a positive result was obtained with the DPRA (Ref. 7.4.1 -3) due to depletion of Cystein peptide despite precipitation. No depletion was observed with the lysine peptide, however, due to technical difficulties (phase separation) this result could not be taken into account. Conducting an h-CLAT is not suitableas a negative result is not accepted for substances with a log Pow> 3.5, what applies to the test item.

In vivo tests are available for the synthetic racemic (+/-)-α-Bisabolol, which is proposed for read across as it contains the test item to 50%:

In a Buehler (Ref. 4.7.1 -1) test according to OECD 406, (+/-)-α-Bisabolol was negative when tested at a concentration of 100% (induction and challenge). In contrast, (+/-)-α-Bisabolol (source substance) was positive when tested in a Guinea Pig Maximisation Test (Ref. 4.7 .1 -2) according to OECD 406 (Induction: 5% intradermal, 100% topic; Challenge: 100% topic).

However, the registered substance is used specifically in cosmetic leave-on formulations up to a concentration of 1% for sensitive skin (CIR Report on Bisabolol, Int. J. Toxicol. 18, Suppl. 3, 33 -40, 1999) . No sensitizing effects are known from the cosmetic use, which has a long history of safe use. Moreover, no sensitizing potential was observed in a clinical sensitization test (Kligman maximisation protocol) in 25 volunteers treated with a cosmetic formulation with 0.1% Bisabolol (CIR, 1999).

Therefore, based on the comprehensive data base available for (-)-α-Bisabolol and the racemic (+/-)-α-Bisabolol (source compound) it is concluded that (-)-α-Bisabolol has no relevant potential for skin sensitization.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The test item (+/-)-α-Bisabolol is a racemic mixture containing the stereoisomers (+)-α-Bisabolol and (-)-α-Bisabolol. Studies on skin sensitisation were not performed for the target substance (-)-α-Bisabolol due to availability of reliable experimental data for (+/-)-α-Bisabolol. (+/-)-α-Bisabolol is considered to be a suitable read across substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance (+/-)-α-Bisabolol, CAS 515-69-5, which was used in the skin sensitisation study, had a purity of 86.8%. No information on impurities is available. The target substance (-)-α-Bisabolol, CAS 23089-26-1, is one of the two stereoisomers of (+/-)-α-Bisabolol.

3. ANALOGUE APPROACH JUSTIFICATION
Experimental data i.e. a skin sensitisation study was available for (+/-)-α-Bisabolol. (+/-)-α-Bisabolol was tested in a skin sensitisation study according to OECD 406 in guinea pigs. No skins sensitising potential was observed in this study. The information given on (+/-)-α-Bisabolol is considered to be sufficient to cover the required endpoint information for the target substance (-)-α-Bisabolol.

Reason / purpose for cross-reference:

read-across source

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 %

No. with + reactions:

15

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 %

No. with + reactions:

15

Total no. in group:

20

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The test item (+/-)-α-Bisabolol is a racemic mixture containing the stereoisomers (+)-α-Bisabolol and (-)-α-Bisabolol. Studies on skin sensitisation were not performed for the target substance (-)-α-Bisabolol due to availability of reliable experimental data for (+/-)-α-Bisabolol. (+/-)-α-Bisabolol is considered to be a suitable read across substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance (+/-)-α-Bisabolol, CAS 515-69-5, which was used in the skin sensitisation study, had a purity of 86.8%. No information on impurities is available. The target substance (-)-α-Bisabolol, CAS 23089-26-1, is one of the two stereoisomers of (+/-)-α-Bisabolol.

3. ANALOGUE APPROACH JUSTIFICATION
Experimental data i.e. a skin sensitisation study was available for (+/-)-α-Bisabolol. (+/-)-α-Bisabolol was tested in a skin sensitisation study according to OECD 406 in guinea pigs. No skins sensitising potential was observed in this study. The information given on (+/-)-α-Bisabolol is considered to be sufficient to cover the required endpoint information for the target substance (-)-α-Bisabolol.

Reason / purpose for cross-reference:

read-across source

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-09-24 to 2018-12-07

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:

adopted 04 February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

12 January 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. In the present study, the test item was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 222.37 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed sufficient reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.64%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

36.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.4320	0.5340	17.6620	0.5340
STD2	8.7580	0.2670	8.8410	0.2670
STD3	4.3320	0.1335	4.4020	0.1335
STD4	2.1060	0.0667	2.1750	0.0667
STD5	1.0150	0.0334	1.0680	0.0334
STD6	0.4700	0.0167	0.5310	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.0850	0.1566	69.79	69.97	0.22	0.31
	5.0140	0.1545	70.21
	5.0620	0.1559	69.92
Test Item	10.8320	0.3320	35.64	36.14	0.91	2.51
	10.8380	0.3321	35.60
	10.5710	0.3240	37.19

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	6.3980	0.1938	61.65	61.30	0.42	0.69
	6.4360	0.1949	61.42
	6.5340	0.1979	60.83
Test Item	16.8800	0.5102	0.00	0.00	0.00	n.a.
	16.8690	0.5099	0.00
	16.8540	0.5094	0.00

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	18.07	--	--	36.14	Moderate Reactivity	positive
Positive Control	65.38	High Reactivity	positive	69.97	Moderate Reactivity	positive

Interpretation of results:

other: should be considered in the context of integrated approached such as IATA

Conclusions:

In this study under the given conditions the test item showed moderate reactivity towards the cysteine peptide. The test item is considered as positive in this in chemico assay for skin sensitisation.
The data generated with this test should be considered in the context of integrated approaches such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study the test item was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 222.37 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (excluding the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight phase separation was observed for the samples of the test item and the positive control (small droplets) including the co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

Precipitation of the test item with the cysteine peptide peak and phase separation with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic cysteine peptide and no reactivity towards the synthetic lysine peptide. The peptide depletion of the cysteine peptides was > 13.89% (36.14%). Even though precipitates with the cysteine peptide and phase separation with the lysine peptide were observed, a positive result can still be used. Based on the prediction model 2 the test item can be considered as positive in this in chemico assay for skin sensitisation.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed sufficient reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.64%.

The controls confirmed the validity of the study for both, the cysteine and lysine run.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-08 to 2018-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.72 (experiment 1); 4.70 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Remarks:

Cell viability was 0.2% at 62.5 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No significant luciferase induction > 1.5 was found. Therefore, no EC1.5 value could be calculated.

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.03

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Remarks:

Cell viability was 0% at 62.5 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

85.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No significant luciferase induction > 1.5 was found. Therefore, no EC1.5 value could be calculated.

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	95.5	102.3	98.9	4.8
	8.00	98.2	107.3	102.7	6.4
	16.00	102.6	104.8	103.7	1.5
	32.00	104.3	109.2	106.7	3.5
	64.00	97.1	105.8	101.4	6.2
Test Item	0.98	92.1	99.9	96.0	5.5
	1.95	98.0	96.5	97.2	1.0
	3.91	96.3	96.3	96.3	0.0
	7.81	95.2	99.6	97.4	3.1
	15.63	91.1	90.5	90.8	0.4
	31.25	88.0	85.6	86.8	1.7
	62.50	0.2	0.0	0.1	0.1
	125.00	0.1	0.0	0.0	0.1
	250.00	0.1	0.1	0.1	0.0
	500.00	0.1	0.1	0.1	0.0
	1000.00	0.1	0.1	0.1	0.0
	2000.00	0.1	0.1	0.1	0.0

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.24	1.14	1.04	1.14	0.10
	8.00	1.09	1.18	1.16	1.15	0.05
	16.00	1.21	1.34	1.39	1.31	0.09
	32.00	1.41	1.75	1.48	1.55	0.18	*
	64.00	2.70	2.77	2.77	2.75	0.04	*
Test Item	0.98	1.27	1.10	1.29	1.22	0.10
	1.95	1.09	1.01	1.08	1.06	0.04
	3.91	1.01	1.03	1.04	1.03	0.01
	7.81	1.02	0.99	1.20	1.07	0.11
	15.63	0.97	1.04	0.99	1.00	0.03
	31.25	1.07	1.04	1.13	1.08	0.04
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.01	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p < 0.05

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.11	1.09	1.02	1.08	0.05
	8.00	1.21	1.30	1.21	1.24	0.06
	16.00	1.20	1.23	1.37	1.27	0.09
	32.00	1.54	1.58	1.59	1.57	0.02	*
	64.00	2.45	2.25	1.84	2.18	0.31	*
Test Item	0.98	1.02	1.04	1.05	1.04	0.01
	1.95	0.95	1.01	0.94	0.97	0.04
	3.91	1.33	1.12	0.99	1.15	0.17
	7.81	1.06	0.94	1.03	1.01	0.06
	15.63	1.05	0.93	1.05	1.01	0.07
	31.25	1.14	0.97	0.97	1.03	0.10
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p < 0.05

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.14	1.08	1.11	0.04
	8.00	1.15	1.24	1.19	0.07
	16.00	1.31	1.27	1.29	0.03
	32.00	1.55	1.57	1.56	0.02	*
	64.00	2.75	2.18	2.46	30.4	*
Test Item	0.98	1.22	1.04	1.13	0.13
	1.95	1.06	0.97	1.02	0.07
	3.91	1.03	1.15	1.09	0.08
	7.81	1.07	1.01	1.04	0.04
	15.63	1.00	1.01	1.00	0.01
	31.25	1.08	1.03	1.05	0.04
	62.50	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.02
	250.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00
	2000.00	29.270.00	0.00	0.00	0.00

* = significant induction according to Student’s t-test, p < 0.05

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n.a.	n.a	n.a.	n.a.
Imax	1.22	1.15	1.18	0.05
IC30[µM]	37.65	36.94	37.30	0.51
IC50[µM]	44.77	44.24	44.51	0.38

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	10.8	pass	10.8	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	± 2 x SD of historical mean	28.87	pass	28.26	pass
Induction PC at 64 µM	2.00 < x < 8.00	2.75	pass	2.18	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.5	96
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.4	0.6	96
EC1.5 PC	7 < x < 34 µM	18.5	6.0	96
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.5	96

Interpretation of results:

other: should be considered in the context of integrated approached such as IATA

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not 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.

Executive summary:

In the present study the test material was dissolved in DMSO.

Based on a molecular weight of 268.5 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first and second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data are considered sufficient to conclude on skin sensitisation potential of the test item under Regulation 1272/2008.

Based on the data available, the substance is not classified for skin sensitisation under Regulation (EC) No 1272/2008.