cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2021 to 21 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

adopted: June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

Test material information

Constituent 1

Reference substance name:

D-Glucitol, anhydro-1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acyl] derivs.

Molecular formula:

C19H37NO5 C21H41NO5

IUPAC Name:

D-Glucitol, anhydro-1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acyl] derivs.

Test material form:

semi-solid (amorphous): gel

Specific details on test material used for the study:

Name: cyclic Glucamide C12-C14
Chemical Name: anhydro-D-glucitol, 1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acryl] derivs.; Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated
Product Name: cyclic N-dodecanoyl/tetradecanoyl-N-methylglucamine / cyclic Glucamide C12-C14
Batch No.: S148866
CAS No: not specified by the sponsor
Molecular Weight: main compounds:
359.5 g/mol (C12-derivative)
387.5 g/mol (C14-derivative)
Calculated Mean Molecular Weight: 314.35 g/mol
Density: 1.07 g/cm3
Purity: 85.90% (based on the calculated mean molecular weight)
Physical State: slightly turbid solid mass
Colour: brown
Stability: stable
Storage Conditions: room temperature
Expiry Date: 30 September 2022
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001886811, 000192647). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube. A factor of 1.16 to correct for the purity of the test item was used. Vortex mixing was used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls

Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
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

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

LUCIFERASE ACTIVITY MEASUREMENTS
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

DATA EVALUATION
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed.

For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.

The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration

Prediction Model
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required.

In addition, a negative result obtained with test items at a maximal test concentration < 1000 µM and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration is considered as inconclusive.

A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

valid

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

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

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

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

Outcome of the prediction model:

negative [in vitro/in chemico]

Any other information on results incl. tables

Cytotoxicity

Table 1: 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	123.1	85.1	104.1	26.9
	8.00	113.8	83.1	98.5	21.7
	16.00	128.8	90.5	109.7	27.1
	32.00	140.2	88.7	114.4	36.4
	64.00	145.4	84.0	114.7	43.4
Test Item	0.98	108.4	98.0	103.2	7.4
	1.95	110.8	107.1	109.0	2.6
	3.91	108.4	104.3	106.3	2.9
	7.81	101.0	100.7	100.8	0.2
	15.63	109.0	97.0	103.0	8.5
	31.25	105.7	79.8	92.7	18.3
	62.50	0.7	36.7	18.7	25.5
	125.00	0.8	18.0	9.4	12.1
	250.00	0.9	4.7	2.8	2.7
	500.00	0.5	0.6	0.5	0.1
	1000.00	0.8	0.6	0.7	0.1
	2000.00	0.9	0.7	0.8	0.1

 

 

 

 

 

 

  Luciferase Activity Experiment 1

Table 2: 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	1.50	0.95	1.25	1.23	0.27
	8.00	1.28	1.24	1.32	1.28	0.04
	16.00	1.43	1.41	1.39	1.41	0.02
	32.00	1.63	1.73	1.81	1.72	0.09	*
	64.00	1.99	2.33	1.83	2.05	0.25	*
Test Item	0.98	1.01	1.19	1.17	1.12	0.10
	1.95	1.08	1.36	1.24	1.23	0.14
	3.91	1.05	1.44	1.19	1.23	0.20
	7.81	1.09	1.34	1.36	1.26	0.15
	15.63	1.15	1.51	1.05	1.24	0.24
	31.25	1.15	1.52	1.40	1.36	0.19
	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

  Luciferase Activity Experiment 2

Table 3: 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	1.42	1.35	0.99	1.25	0.23
	8.00	1.57	1.43	1.30	1.43	0.14
	16.00	1.28	1.42	1.75	1.49	0.24
	32.00	1.58	1.86	2.09	1.85	0.26	*
	64.00	7.50	6.04	6.41	6.65	0.76	*
Test Item	0.98	0.93	0.81	0.96	0.90	0.08
	1.95	0.90	0.91	0.96	0.92	0.03
	3.91	1.02	1.09	1.00	1.04	0.04
	7.81	1.03	0.93	1.06	1.00	0.07
	15.63	1.24	1.23	1.72	1.39	0.28
	31.25	1.31	1.46	1.39	1.39	0.07
	62.50	0.03	0.02	0.10	0.05	0.04
	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

Luciferase Activity - Overall Induction

Table 4: Induction of Luciferase Activity – Overall Induction

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	1.23	1.25	1.24	0.01
	8.00	1.28	1.43	1.36	0.10
	16.00	1.41	1.49	1.45	0.05
	32.00	1.72	1.85	1.79	0.09	*
	64.00	2.05	6.65	4.35	3.25
Test Item	0.98	1.12	0.90	1.01	0.16
	1.95	1.23	0.92	1.07	0.21
	3.91	1.23	1.04	1.13	0.14
	7.81	1.26	1.00	1.13	0.18
	15.63	1.24	1.39	1.32	0.11
	31.25	1.36	1.39	1.37	0.02
	62.50	0.00	0.05	0.03	0.03
	125.00	0.00	0.00	0.00	0.00
	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	0.00	0.00	0.00	0.00

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

Additional Parameters

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5	n.a.	n.a.	n.a.	n.a.
Imax	1.36	1.39	1.38	0.03
IC30	41.87	38.37	40.12	2.48
IC50	47.83	52.88	50.35	3.57

n.a.=not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

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.

Executive summary:

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.

In the present study cyclic Glucamide C12-C14 was dissolved in DMSO.

Based on a calculated molecular weight of 314.35 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 experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 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.

The controls confirmed the validity of the study