Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 - 22 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 04 February 2015

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 25 June 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

EC Number:

947-918-6

Molecular formula:

not applicable, UVCB substance.

IUPAC Name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

Test material form:

solid

In vitro test system

Details on the study design:

TEST SYSTEM:
- KeratinoSens™: immortalised adherent cell line derived from HaCaT human keratinocytes transfected with a stable insertion of the Luciferase construct

TEST SAMPLE PREPARATION:
The test item was dissolved in tetrahydrofuran (THF) (purity ≥ 99%). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium. Vortex mixing was used to aid solubilisation.

Based on the stock solution a set of 12 master solutions in 100% solvent was prepared. The stock solution of the test item was diluted 11x 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. Since the test item was dissolved in THF, DMSO was added at a final concentration of 4% (v/v). 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.

CONTROLS:
- Blank: A blank well with no seeded cells was included in every plate to determine the background.
- Negative Control: DMSO at a final concentration of 1% (v/v) in test item exposure medium
- Solvent Control: THF at a final concentration of 1% (v/v) in test item exposure medium
- Positive Control: Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; > 98%). CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

CELL LINE:
Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number < 25 (P 2 in experiment 1; P 10 in experiment 2) were used. Cells were cultured in 75 cm2 culture flasks in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator.

MEDIA:
- Maintenance Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate, supplemented with 10% fetal bovine calf serum (FBCS) and 1% geneticin.
- Assay Mediuim: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate, supplemented with 10% fetal bovine calf serum (FBCS)
- Test Item Exposure Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate, supplemented with 1% fetal bovine calf serum (FBCS)

DOSE GROUPS:
- Negative Control: 1% (v/v) DMSO in test item exposure medium and 1% (v/v) THF
- Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM
- Test Item: 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.

EXPERIMENTAL PRODECURE:
For the test item two independent experiments using separately prepared test item solutions and independently harvested cells were conducted. Each independent run consisted of three replicates for every concentration step of the test item and the positive control.

A cell suspension of 8 × 10E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 10E4 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 Dulbecco’s Phosphate Buffered Saline (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. 50 μL of the luciferase substrate per well were injected by the injector of the plate reader. The plate reader waited for 1 s before assessing the luciferase activity for 2 s. 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% Sodium Dodecyl Sulphate (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 over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the Optical Density (OD) was measured at λ = 600 nm.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Fold Induction of luciferase activity

Value:

0.83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Fold Induction of luciferase activity

Value:

0.91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
Refer to section "Any other information on results incl. tables" below.

Any other information on results incl. tables

 

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	102.3	105.8	104.0	2.4
	8.00	108.3	99.1	103.7	6.5
	16.00	114.1	112.3	113.2	1.3
	32.00	116.8	118.6	117.7	1.3
	64.00	112.1	122.5	117.3	7.3
Test item	0.98	103.4	108.5	105.9	3.5
	1.95	105.8	103.7	104.7	1.5
	3.91	104.9	97.8	101.3	5.0
	7.81	96.5	92.8	94.7	2.6
	15.63	101.1	96.4	98.8	3.3
	31.25	107.6	99.9	103.7	5.4
	62.50	105.8	102.5	104.1	2.3
	125.00	114.3	113.5	113.9	0.6
	250.00	122.0	119.1	120.6	2.1
	500.00	131.3	117.4	124.3	9.8
	1000.00	119.2	116.6	117.9	1.8
	2000.00	110.2	110.5	110.3	0.2

 

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	0.95	0.99	1.22	1.05	0.15
	8.00	1.13	1.30	1.30	1.24	0.10
	16.00	1.43	1.68	1.79	1.64	0.18	YES
	32.00	2.25	2.59	2.85	2.56	0.30	YES
	64.00	5.65	7.51.	7.99	7.05	1.24	YES
Test item	0.98	1.36	1.34	1.51	1.41	0.09
	1.95	1.10	0.81	0.87	0.93	0.16
	3.91	0.98	0.96	0.84	0.93	0.08
	7.81	1.07	1.06	0.98	1.04	0.05
	15.63	0.86	0.81	0.82	0.83	0.03
	31.25	1.04	0.94	0.95	0.98	0.05
	62.50	0.98	0.94	0.95	0.96	0.02
	125.00	1.17	0.85	1.02	1.01	0.16
	250.00	1.36	1.07	1.10	1.18	0.16
	500.00	1.27	1.05	1.36	1.22	0.16
	1000.00	1.32	1.06	1.15	1.18	0.13
	2000.00	1.48	1.25	1.15	1.30	0.17

 

Table 3: Induction of Luciferase Activity Experiment 2

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	1.21	1.28	1.49	1.32	0.15
	8.00	1.11	1.37	1.29	1.26	0.13
	16.00	1.15	1.44	1.35	1.32	0.15
	32.00	1.83	1.70	2.13	1.88	0.22	YES
	64.00	3.77	3.63	3.97	3.79	0.17	YES
Test item	0.98	1.080	1.17	0.99	1.08	0.09
	1.95	0.84	1.02	0.88	0.91	0.09
	3.91	0.89	0.92	1.00	0.94	0.05
	7.81	0.96	1.09	0.92	0.99	0.09
	15.63	0.87	0.97	0.95	0.93	0.06
	31.25	0.90	0.99	1.01	0.97	0.06
	62.50	0.92	0.99	0.97	0.96	0.03
	125.00	1.03	1.15	1.22	1.13	0.09
	250.00	1.02	1.26	1.28	1.19	0.14
	500.00	1.08	1.55	1.21	1.28	0.24
	1000.00	1.08	1.10	1.11	1.10	0.01
	2000.00	0.85	1.13	1.12	1.04	0.16

 

Table 4: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	14.6	pass	9.3	pass
CV Solvent Control TI (1% THF)	< 20%	20.0	pass	9.6	pass
No. of positive control concentration steps with significant luciferase activity induction > 1.5	≥ 1	3.0	pass	2.0	pass
EC1.5 positive control	7 < x < 34 µM	13.22	pass	21.19	pass
Induction positive control at 64 μM	2.00 < x < 8.00	7.05	pass	3.79	pass

 

Table 5: Historical Data

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 positive control	7 < x < 34 µM	18.5	6.0	96
Induction positive control at 64 μM	2.00 < x < 8.00	3.8	1.5	96

 

Applicant's summary and conclusion

Interpretation of results:

other: negative for keratinocyte activation

Conclusions:

After 48 h of exposure to the test substance luciferase activity in KeratinoSens™ cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. 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. From this it has to be concluded that the test substance has no keratinocyte activating potential.

Executive summary:

negative for keratinocyte activation