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

Endpoint summary

Description of key information

In-vitro/Chemico skin sensitisation:

DPRA (OECD 442C): negative

KeratinoSens (OECD 442D): negative

h-CLAT (OECD 442E): negative

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:

key study

Study period:

27 April 2021 to 21 October 2021

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 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

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.

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]

Positive control results:

valid

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.

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]

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

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

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2021 to 14 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

23rd July 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

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)
Density: 1.07 g/cm3
Purity: 100% (UVCB)
Physical State: slightly turbid solid mass
Colour: brown
Log KOW: not specified by the sponsor
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.

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test
item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely
dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in
the human monocytic cell line THP-1. The expression of the cell surface markers compared to the
respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both
experiments. The threshold of 150% for CD86 (150% experiment 1; 266% experiment 2) and
200% for CD54 (388% experiment 1; 356% experiment 2) were clearly exceeded.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD54>200

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

All CD54 was not upregulated above the threshold of 200%

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD86>150

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

All CD86 was not upregulated above the threshold of 150%

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI CD86>150

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

All CD86 was not upregulated above the threshold of 150%

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI CD54 > 200

Value:

1

Cell viability:

12.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: RFI=205 at Concentration: 62.47 µg/mL (Cell viability 12.3). At other dose levels, CD54 was not upregulated above the threshold of 200%

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Results of the Cell Batch Activation Test (Batch 11)

 

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	86	265	>150	87	393	>200	yes	pass
NiSO4	100 µg/mL	83	219	>150	79	711	>200	yes	pass
LA	1000 µg/mL	91	88	≤150	92	111	≤200	no	pass

 

Results of the Cell Batch Activation Test (Batch 12)

 

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	89	349	>150	88	386	>200	yes	pass
NiSO4	100 µg/mL	97	244	>150	89	1007	>200	yes	pass
LA	1000 µg/mL	88	125	≤150	94	181	≤200	no	pass

 

 

Results of the Dose Finding Assay

 

Sample		Experiment 1		Experiment 2
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	93.38	--	92.83
Solvent Control	DMSO	--	93.97	--	94.25
cyclic Glucamide C12-C14	C8	7.81	93.07	7.81	88.70
	C7	15.63	93.39	15.63	91.77
	C6	31.25	91.75	31.25	89.70
	C5	62.50	70.65	62.50	67.96
	C4	125.00	2.30	125.00	2.70
	C3	250.00	2.53	250.00	2.53
	C2	500.00	1.27	500.00	1.64
	C1	1000.00	3.78	1000.00	4.44
Calculated CV75 [µg/mL]		54.18		49.93
Mean CV75 [µg/mL]		52.06
SD CV 75 [µg/mL]		3.00

 

CD54 and CD86 Expression Experiment 1

 

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio [%] Isotype IgG1 to:
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.0	92.8	92.9	1672	713	364	1308	349	88	90	459	196
Solvent Control	0.20%	93.9	93.8	93.6	1828	728	340	1488	388	100	100	538	214
DNCB	4.00	76.2	74.8	75.1	2598	1869	364	2234	1505	150	388	714	513
cyclic Glucamide C12-C14	62.47	6.4	12.3	7.7	691	1117	320	371	797	25	205	216	349
	52.06	51.4	49.2	58.4	696	986	337	359	649	24	167	207	293
	43.38	65.2	67.2	67.9	1027	1007	352	675	655	45	169	292	286
	36.15	84.6	82.7	84.1	1312	912	341	971	571	65	147	385	267
	30.13	90.1	90.3	90.4	1298	743	329	969	414	65	107	395	226
	25.11	89.6	91.2	91.5	1324	689	320	1004	369	67	95	414	215
	20.92	91.1	92.4	93.1	1441	662	314	1127	348	76	90	459	211
	17.43	91.3	92.3	91.5	1317	663	350	967	313	65	81	376	189

 

 

 

 

CD54 and CD86 Expression Experiment 2

 

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio [%] Isotype IgG1 to:
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.3	93.9	93.4	1933	622	474	1459	148	104	73	408	131
Solvent Control	0.20%	94.0	93.1	92.2	1838	639	436	1402	203	100	100	422	147
DNCB	4.0	82.4	85.5	85.6	4219	1214	491	3728	723	266	356	859	247
cyclic Glucamide C12-C14	62.47	28.1	27.7	33.1	714	790	440	274	350	20	172	162	180
	52.06	47.8	51.3	52.9	963	789	401	562	388	40	191	240	197
	43.38	70.8	70.2	73.8	1241	742	401	840	341	60	168	309	185
	36.15	84.4	84.5	86.4	1439	681	398	1041	283	74	139	362	171
	30.13	88.9	91.5	86.7	1317	603	391	926	212	66	104	337	154
	25.11	91.2	90.9	92.1	1468	556	383	1085	173	77	85	383	145
	20.92	90.2	93.6	91.2	1449	594	378	1071	216	76	106	383	157
	17.43	91.7	90.6	91.5	1535	586	391	1144	195	82	96	393	150

 

Acceptance Criteria

 

Acceptance Criterion	range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability medium and solvent control [%]	>90	92.8	-	94.0	pass	92.2	-	96.3	pass
number of test dosed with viability >50% CD86	≥4	7			pass	6			pass
number of test dosed with viability >50% CD54	≥4	6			pass	7			pass
number of test dosed with viability >50% IgG1	≥4	7			pass	7			pass
RFI of positive control of CD86	≥150	150			pass	266			pass
RFI of positive control  of CD54	≥200	388			pass	356			pass
RFI of solvent control of CD86	<150	114			pass	96			pass
RFI of  solvent control  of CD54	<200	111			pass	137			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	459			pass	408			pass
MFI ratio CD86/IgG1 for solvent control [%]	>105	538			pass	422			pass
MFI ratio CD54/IgG1 for medium control [%]	>105	196			pass	131			pass
MFI ratio CD54/IgG1 for solvent control [%]	>105	214			pass	147			pass

 

 

Historical Data

 

Criterion	mean	SD	N
cell viability solvent controls [%]	96.3	1.5	1566
number of test doses with viability >50%	-	-	4058
RFI of positive control of CD86	354.4	122.7	261
RFI of positive control of CD54	434.5	254.7	261
RFI of solvent control of CD86	111.0	31.2	261
RFI of solvent control of CD54	114.5	43.1	261
MFI ratio IgG1/CD86 for medium control [%]	308.5	153.7	261
MFI ratio IgG1/CD86 for DMSO control [%]	352.3	362.9	261
MFI ratio IgG1/CD54 for medium control [%]	179.0	149.7	261
MFI ratio IgG1/CD54 for DMSO control [%]	176.6	54.1	261

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in two independent experiment runs. Therefore, the test item is 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:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers 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. For the dose finding assay stock solutions with concentrations ranging from 500.00 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

 

A CV75 of 52.06 ± 3.00 µg/mL was derived in the dose finding assay

 

Based on the CV75, the main experiment was performed covering the following concentration steps:

62.47, 52.06, 43.38, 36.15, 30.12, 25.10, 20.92 and 17.43 µg/mL

 

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

 

Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 6.4% (CD86), 12.3% (CD54) and 7.7% (isotype IgG1 control) in the first experiment and to 28.1% (CD86), 27.7% (CD54) and 33.1% (isotype IgG1 control) in the second experiment.

 

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was upregulated above the threshold of 200% in first experiment at the highest test item concentration. As viability was <50% (12.3%) at the highest test item concentration, the concentration was excluded from further evaluation. In the second experiment, the expression of cell surface marker CD54 was not upregulated above the threshold of 200%. Therefore, the test item can be considered as non-sensitiser.

 

The controls confirmed the validity of the study for all experiments.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-04 to 2021-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

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.

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.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.65%.

Key result

Group:

test chemical

Run / experiment:

other: cysteine run (Test Item (100mM))

Parameter:

cysteine depletion

Value:

2.89

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: cysteine run (Test Item (maximum solubility))

Parameter:

cysteine depletion

Value:

5.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

other: lysine run (Test Item (100 mM))

Parameter:

other: mean peptide depletion [%]

Value:

2.26

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: lysine run (Test Item (maximum solubility))

Parameter:

other: mean peptide depletion [%]

Value:

1.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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	17.0730	0.5340	15.4430	0.5340
STD2	8.6330	0.2670	7.8040	0.2670
STD3	4.3570	0.1335	3.9380	0.1335
STD4	2.1380	0.0667	1.9680	0.0667
STD5	1.0580	0.0334	1.0020	0.0334
STD6	0.4820	0.0167	0.5040	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	4.8680	0.1517	69.47	69.73	0.41	0.59
	4.8600	0.1514	69.52
	4.7500	0.1480	70.21
Test Item (100 mM)	15.6970	0.4896	0.10	2.89	3.72	128.78
	15.4840	0.4829	1.46
	14.5940	0.4551	7.12
Test Item (maximum solubility)	15.0150	0.4683	4.44	5.54	1.09	19.76
	14.8410	0.4628	5.55
	14.6710	0.4575	6.63

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	5.4140	0.1860	62.63	61.56	1.28	2.08
	5.5180	0.1896	61.91
	5.7750	0.1984	60.14
Test Item (100 mM)	14.4100	0.4971	2.07	2.26	0.48	21.35
	14.3000	0.4933	2.81
	14.4330	0.4979	1.91
Test Item (maximum solubility)	14.5540	0.5021	1.09	1.69	0.74	43.97
	14.3440	0.4948	2.51
	14.5000	0.5002	1.45

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 (100 mM)	2.58	Minimal Reactivity	negative	2.89	Minimal Reactivity	negative
Test Item (maximum solubility)	3.61	Minimal Reactivity	-	5.54	Minimal Reactivity	-
Positive Control	65.65	High Reactivity	positive	69.73	Moderate Reactivity	positive

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the 100 mM test item solution showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.
In this study under the given conditions the maximum solubility solution of the test item showed minimal reactivity towards both peptides. Due to the observed turbidity the prediction model does not apply and a prediction cannot be made for the maximum solubility solution of the test item.
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 cyclic Glucamide C12-C14 was dissolved in isopropanol, based on the results of the pre-experiments.

Based on a molecular weight of314.35 g/mola 100 mM stock solution was prepared and additionally the neat test item was tested to its maximum solubility. The test item solutions were testedby 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 and the maximum solubility solution of the test item no turbidity or precipitation 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. No precipitation, turbidity or phase separation was observed for the samples of the100 mM stock solution of thetest item. Slight turbidity was observed for the samples of themaximum solubility solution of thetest item (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution and the maximum solubility solution of the test item no turbidity or precipitation 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Slight turbidity was observed for the samples of the positive control (excluding 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 turbidity was regarded as not relevant.

For the 100 mMstock solution of the test item, no co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_isopropanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was£ 6.38% (2.58%).Based on the prediction model 1 the test item can be considered as non-sensitiser.

For themaximum solubility stock solution of thetest item,turbidity in the cysteine experiment was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

Themaximum solubilitystock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was£ 6.38% (3.61%).

According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed turbidity in the cysteine experiment no prediction can be madefor themaximum solubility stock solution of thetest item.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of bothpeptides was 65.65%.

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

In three in-chemico/vitro studies according to OECD 442 C/D/E, no skin sensitisation potential is indicated. Therefore, the resitered substance is considered as non-sensitizer and not classified for skin sensitisation.