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

Endpoint summary

Description of key information

Skin sensitisation (OECD 442D): not activating keratinocytes

Skin sensitisation (OECD 429): not sensitising

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:

14 - 22 June 2018

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)

Version / remarks:

adopted 04 February 2015

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

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.

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

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.

 

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

 

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

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Aug - 11 Sept 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

23 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Inc.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks (preliminary animals), 11 weeks (test and control animals)
- Weight at study initiation: 19.4 - 23.8 g (test and control animals)
- Housing: Individually in plastic solid bottom cages during the dosing and resting phase. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. Enrichment (e.g., nesting material) was placed in each cage.
- Diet: Envigo Teklad Global 16% Protein Rodent Diet® #2016, ad libitum
- Water: Filtered tap water, ad libitum
- Acclimation period: 7 or 21 days
- Indication of any skin lesions: no, only healthy animals without indication of skin lesions were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 43 - 67
- Air changes (per hr): 14
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

other: 1% (w/w) Pluronic® L92

Concentration:

Preliminary toxicity test: 0, 5, 10, and 25%
Main test: 0, 5, 10, and 25%

No. of animals per dose:

Preliminary toxicity test: 2
Main test: 5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Test substance was suspendable in 1% Pluronic® L92. Preliminary sample preparation testing indicated that mixtures containing 50% test substance were too viscous for dosing. Therefore, a 25% concentration of the test substance in 1% Pluronic® L92 was the highest concentration tested.
- Irritation: Prior to each application (Days 1, 2, and 3) and on Day 6, the ears were evaluated for erythema and edema.
- Erythema and edema scores: According to the modified Draize scoring system
- Systemic toxicity: All preliminary mice were observed for signs of mortality, gross toxicity, and/or behavioral changes daily. Preliminary mice were euthanized via CO2 inhalation on Day 6.
- Ear thickness measurements: not performed

MAIN STUDY
- Systemic toxicity: All test and control mice were observed for signs of mortality, gross toxicity, and/or behavioral changes daily. All test and control mice were euthanized via overdose of inhaled lsoflurane anesthetic on Day 6.
- Name of test method: 3H-methyl thymidine incorporation determined by beta-scintillation counting

TREATMENT PREPARATION AND ADMINISTRATION:
Approximately 5 h after the injection of 20 µCi of 3H-methyl thymidine, test and control mice were euthanized and the draining auricular lymph nodes were excised. The lymph nodes were evaluated for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in phosphate buffered saline (PBS) by gently massaging the lymph nodes between the frosted ends of two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuged for approximately 10 minutes at 1800 rpm. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centrifugation. After the second wash, 5 mL of 5% trichloroacetic acid (TCA) in distilled water was added to the sediment and the tube was vortexed briefly. The DNA was then precipitated in the 5% TCA in distilled water at approximately 4 °C overnight (approximately 18 hours). Following the overnight precipitation of the DNA, the tubes were centrifuged again for approximately 10 minutes at 1800 rpm and the supernatant was discarded. The resulting precipitate was re-suspended using 1 mL of 5% TCA in distilled water and transferred to 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by beta-scintillation counting and expressed as disintegrations per minute (dpm), minus background dpm.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was performed on the disintegrations per minute (dpm) values. Significance was judged at p < 0.05. The treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance, followed by comparison of the treated groups to control by Dunnett's t-test for multiple comparisons. Where variances are considered significantly different by Bartlett's test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn's test).

Positive control results:

The positive control substance (25% Hexylcinnamic aldehyde in 1% Pluronic® L92) induced a positive reaction in the animals of the positive control group. The Stimulation Index (SI) of the pooled group was 5.63, well above the threshold of 3 indicating a positive response.

Key result

Parameter:

SI

Value:

1.09

Test group / Remarks:

5% in 1% Pluronic® L92

Key result

Parameter:

SI

Value:

1.11

Test group / Remarks:

10% in 1% Pluronic® L92

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

25% in 1% Pluronic® L92

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The mean and standard deviation of the disintegrations per minute (dpm) values were calculated for each dose group. A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. Any test substance that produces an SI 3 in the LLNA is normally considered positive for dermal sensitisation potential.

EC3 CALCULATION
The EC3 value was not calculated since all dose levels induced a stimulation index of less than 3.0.

CLINICAL OBSERVATIONS, BODY WEIGHTS
All animals appeared active and healthy throughout the study. Seven mice from the test, all mice from the vehicle control, and three from the positive control groups lost body weight during the study. All other mice gained body weight during the study.

SKIN REACTIONS
No dermal irritation was observed for any of the vehicle control group and test group sites in any animal (scores 0 for erythema and edema). In the positive control group, very slight to well-defined erythema (scores of 1 and 2) was evident at eight positive control sites on Day 2, all sites on Day 3, and nine sites on Day 6. Slight edema (score of 1) was present at two sites on Day 3 and four sites on Day 6. Desquamation was present at all sites on Day 6.

 

Table 1: Individual dpm values

Background:	54.09
Group	dpm	dpm minus background^*	Group mean dmp	Std. dev.	SI^**
1 Vehicle control (1%Pluronic® L92)	1301.35	1247-26	1879.07	499.59	--
	1767.79	1713.70
	2426.30	2372.21
	1709.38	1655.29
	2460.96	2406.87
2 Positive control (25% HCA in 1% Pluronic® L92)	16139.84	16085.75	10586.97	3310.86	5.63
	10422.62	10368.53
	10566.32	10512.23
	8101.95	8047.86
	7974.57	7920.48
3 5% Test substancein 1% Pluronic® L92)	2586.05	2531.96	2045.02	458.96	1.09
	1359.63	1305.54
	2239.03	2184.94
	2027.50	1973.41
	2283.36	2229.27
4 10% Test substancein 1% Pluronic® L92)	2753.53	2699.44	2088.44	527.67	1.11
	2278.87	2224.78
	1304.47	1250.38
	2275.50	2221.41
	2100.26	2046.17
5 25% Test substancein 1% Pluronic® L92)	2627.66	2618.57	2245.98	318.29	1.20
	2458.66	2404.57
	1883.57	1829.48
	2417.68	2363.59
	2067.77	2013.68

dpm: disintegrations per minute

^*: Values analyzed for outliers

^**: Stimulation Index= Average dpm of Test Substance/Average dpm of Vehicle

 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Based on these findings and on the evaluation system used, the test substance is not considered to be a contact dermal sensitiser at concentrations less than or equal to 25% in the LLNA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

General considerdations

According to Annex VII, item 8.3.1., of the REACH Regulation (EC) No. 1907/2006, information allowing a conclusion whether the substance is a skin sensitiser and whether it can be presumed to have the potential to produce significant sensitisation in humans must be provided by means of in vitro / in chemico methods. Since the in vitro / in chemico methods have certain restrictions with respect to, e.g., the Log Pow values of substances that can be tested or are not applicable to UVCB substances, interpretation of their results must be made with care. In case no unambigous and/or reliable result can be obtained, in vivo testing for skin sensitisation is warranted.

In vitro / in chemico studies

In a study according to OECD guideline 442D, performed under GLP conditions (Rödig, 2018a), the skin sensitisation potential of the registered substance was investigated. 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. The test substance was dissolved in THF and 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 the study the test item is considered as not activating keratinocytes.

It must be noted that the Log Pow value of the test substance is > 7 and the test guideline applied (OECD guideline 442D, adopted 04 February 2015) does not yield reliable results for substances with Log Pow > 7. However, the OECD 442D test guideline has been updated in the meantime and its most recent version has been adopted 25 June 2018. The most current guideline version states with regard to the chemical nature of the test substance: "In genera mono constituent substances with a LogP above 7 may be insoluble in the exposure medium, however, if solubility or stable dispersion can be obtained and documented, testing may still be conducted." UVCB and multi-constituent substances are not explicitly excluded as long as stable solutions of the test item with proper concentrations can be obtained. Since this was possible with the registered substance, the study is considered valid.

In addition, Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols was aslo investigated in an in vitro human cell line activation test (h-CLAT) according to OECD guideline 442E and observing GLP principles (Rödig, 2018b). Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing. In the study the test substance was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared. 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. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps: 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 μg/mL In the dose-finding experiment precipitation was observed at the two highest test item concentrations when mixing the test item stock solutions with cell culture medium. In the main experiments precipitation 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. No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 94.4% (CD86), 94.1% (CD54) and 93.8% (isotype IgG1 control) in the first experiment and to 92.0% (CD86), 91.6% (CD54) and 91.7% (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 not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered not to activate dendritic cells. However, in the main experiment no cytotoxic effects but signs of precipitates were observed in all test item concentrations. An interaction between the test item and the cells could not be guaranteed and, hence, the reliability of the study cannot be guaranteed. In conclusion, the result of this assay is considered as supportive information only and no hazard conclusion can be derived from the assay.

In vivo study

No final hazard assessment and no reliable derivation of the classification of the registered substance can be achieved based on the two in vitro / in chemico assays reported as the h-CLAT test turned out to be not reliable. In order to overcome the deficiencies of the in vitro / in chemico investigations and to reliably conclude on the skin sensitisation hazard, the skin sensitisation potential of Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols has been tested using the Local Lymph Node Assay (LLNA) according to OECD guideline 429 under GLP conditions (Lowe, 2018b). Three concentrations of the test substance (5%, 10% and 25%) in 1% Pluronic® L92 Surfactant in distilled water and the vehicle alone were topically applied to twenty healthy female CBA:J mice (five mice/group) for three consecutive days. Three days after the last  application, the mice were given an i.v. injection containing 20 µCi of 3H-methyl thymidine. Approx. five hours later, all animals were euthanized via an overdose of inhaled Isoflurane and the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. Each animal's ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the i.v. injection. A positive control group (five animals) was treated with a 25% w/w mixture of alpha-Hexylcinnamaldehyde (HCA) in 1% Pluronic® L92 in the same manner as the test animals. Stimulation indices of 5.63, 1.09, 1.11, and 1.20 were determined for the positive control, 5% test item, 10% test item and 25% test item groups, respectively. The EC3 value was not calculated since all dose levels induced a stimulation index of less than 3.0. All animals appeared active and healthy throughout the study. Seven mice from the test, all mice from the vehicle control, and three from the positive control groups lost body weight during the study. All other mice gained body weight during the study. No dermal irritation was observed for any of the vehicle control group and test group sites in any animal (scores 0 for erythema and edema), while in the positive control group, very slight to well-defined erythema (scores 1 and 2) and slight edema (score 1) was evident. Based on the results of this study, the test substance is not considered to be a contact dermal sensitiser at concentrations less than or equal to 25% in the LLNA. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are, therefore, conclusive but not sufficient for classification.