Reaction product of 2,2'-oxydiethanol and 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate and hexan-6-olide and trimethylhexa-1,6-diyl diisocyanate

Endpoint summary

Description of key information

Skin sensitisation in vitro (OECD 422D): positive for keratinocyte activation

Skin sensitisation in vitro (OECD 442E): positive for activation of dendritic cells

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:

weight of evidence

Study period:

30 Jan - 09 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (in vitro Skin Sensitisation: human Cell Line Activation Test)

Version / remarks:

adopted 25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of study:

activation of dendritic cells

Details on the study design:

TEST METHOD:
The in vitro human Cell Line Activation Test (h-CLAT) is an alternative testing method for the evaluation of the skin sensitisation potential of a test compound. It quantifies phenotypic changes, such as cell surface marker expression in cell lines following 24 h treatment with chemicals. The human leukemia cell line THP-1 is used as surrogate for human myeloic dendritic cells, which show enhanced CD86 and CD54 surface protein expression when treated with sensitisers.

TESTS SUBSTANCE PREPARATION:
The test item was dissolved in dimethyl sulfoxide (DMSO).

CONCENTRATIONS:
Pre-experimental dose-finding study:
first run: 8, 16, 31, 63, 125, 251, 501 and 1003 µg/mL
second run: 18, 21, 25, 30, 37, 44, 53 and 63 µg/mL
Main experiment (h-CLAT): based on the results obtained in the pre-experimental dose-finding study: 15, 18, 22, 26, 31, 38, 45 and and 54 µg/mL.

VEHICLE CONTROL: 0.2% DMSO in Roswell Park Memorial Institute (RPMI) medium
POSITIVE CONTROL CV75: 1-chloro-2,4-dinetrobenzene (DNCB) prepared as 4.2 µg/mL in DMSO
POSITIVE CONTROL CD54 and CD86 expression: Nickel Sulphate prepared as 100 µg/mL in RPMI medium

TEST CELL LINE: THP-1 cells
- Source: ATCC (LGC Standards GmbH, Germany Office), #TIB-202
- Passage number 5 to 8

CELL CULTURE CONDITIONS:
- Type and identity of media: Complete RPMI-1640 culture medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.05 mM L-glutamine and 0.05 mM 2-mercaptoethanol
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5 ± 0.5
- Seeding h-CLAT test: 0.9-1 x 10E6 cells per well in 24-well format, total volume 550 µL

EXPOSURE CONDITIONS:
- Method of application: in medium
- Exposure duration: 24 ± 0.5 h

NUMBER OF REPLICATES: Each concentration was tested in three independent runs

DETERMINATION OF CYTOTOXICITY:
- Method: Propidium iodide uptake, 24 ± 0.5 h exposure with test item
- Detection: Flow cytometry, Apogee Flow Cytometer
- Determination of cell viability: calculation of the CV75, which corresponds to the concentration needed to reduce the relative absorbance to 75% of the solvent control.

DETERMINATION OF FLUORESCENCE:
- Flow cytometry, Apogee Flow Cytometer
- Antibodies: fluorescein isothiocyanate labelled CD86 and CD54

Positive control results:

Relative fluorescence intensities first experiment:
4.2 µg/mL: CD54 = 514%, CD86 =520%

Relative fluorescence intensities, second experiment:
4.2 µg/mL: CD54 = 489%, CD86 = 378%

Relative fluorescence intensities, third experiment:
4.2 µg/mL: CD54 = 781%, CD86 = 570%

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in 2/3 independent runs

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: RFI in % for CD54 in µg/mL

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in 3/3 independent runs

Remarks:

cell viability was < 50% in the third run

Other effects / acceptance of results:

OTHER EFFECTS:
Cell viability at the highest dose in the third run was < 50%, therefore the corresponding RFI value was not considered valid and was excluded from the prediction. All other cell viabilities complied with the acceptance criteria.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, solvent control RFI values do not exceed the positive criteria CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%
- Acceptance criteria met for positive control: yes, RFI values CD86 ≥ 150% and CD54 ≥ 200% and cell viability is > 50%

Table 2: Results of the hCLAT test, first experiment

Compound	Concentration [µg/mL]	RFI CD86	RFI CD54	Cell viability [%]
control		100	100	91.6
DMSO	0.20%	59	96	93.5
DNCB	4.2	520	514	74.2
Test item	54	104	39	22.4
	45	149	112	44.4
	38	88	164	74.8
	31	121	117	82.1
	26	132	127	87.1
	22	88	130	88.5
	18	148	201	89.8
	15	161	102	92.3

Table 3: Results of the hCLAT test, second experiment

Compound	Concentration [µg/mL]	RFI CD86	RFI CD54	Cell viability [%]
control		100	100	92.1
DMSO	0.20%	121	88	92.9
DNCB	4.2	379	489	76.2
Test item	54	59	90	20.3
	45	94	100	27.8
	38	97	163	70.5
	31	89	268	81.8
	26	105	228	84.0
	22	63	174	88.7
	18	95	161	85.8
	15	52	125	89.9

Table 4: Results of the hCLAT test, third experiment

Compound	Concentration [µg/mL]	RFI CD86	RFI CD54	Cell viability [%]
control		100	100	91.4
DMSO	0.20%	97	119	93.4
DNCB	4.2	570	781	58.9
Test item	54	114	154	10.0
	45	199	347	39.3
	38	227	334	66.0
	31	200	387	85.2
	26	208	260	84.4
	22	183	400	83.0
	18	214	207	86.2
	15	142	101	90.6

Interpretation of results:

other: positive for activation of dendritic cells

Conclusions:

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Feb - 22 Mar 2019

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 25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of study:

activation of keratinocytes

Details on the study design:

TEST CELL LINE
- Cell type: HaCaT cells (human keratinocytes)
- Source: Givaudan, Switzerland
- Passage number: < 25

TEST METHOD
The in vitro KeratinoSensTM assay enables the detection of the skin sensitizing potential of a test item by analysing the activation of keratinocytes. This activation step represents the second molecular key event of the adverse outcome pathway, which is the induction of cyto-protective signaling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSensTM assay addresses the effect on the antioxidant response element (ARE) Nrf2-dependent pathway in the transgenic KeratinoSensTM cell line, which stably expresses the ARE-Nrf2-dependet luciferase gene.

TEST SUBSTANCE PREPARATION
All test item solutions were freshly prepared prior to use. The test substance was dissolved in dimethyl sulfoxide (DMSO) preparing a stock solution of 40 mg/mL (200 mM), which was further used for a dilution series of 12 x dilution 1:5 in DMSO. These 100x concentrated master solutions were further diluted 1:25 with cell culture medium.

CONCENTRATIONS:
Experiments 1 and 2: 1, 2, 4, 8, 16, 31, 63, 125, 250, 500, 1000 and 2000 µM
Experiment 3: 0.7, 1.1, 1.6, 2.4, 3.7, 5.5, 8.2, 12.3, 18.5, 27.8, 41.7 and 62.5 µM

CONTROLS:
A blank, a negative and a positive control were included.
BLANK CONTROL: A blank well with no seeded cells was included in every plate to determine the background. This well remained untreated.

NEGATIVE CONTROL: Dimethyl sulfoxide (DMSO), Merck; Lot no.: STBH8906 at a final concentration of 1% (v/v) in test item exposure medium. The negative control is actually a vehicle control.

POSITIVE CONTROL: trans-Cinnamaldehyde (CA); Sigma; Lot no. STBG0250V; CAS 14371-10-9; purity 99.1% used at a final concentration range of 4 - 64 µM.

LUCIFERASE ASSAY SYSTEM:
- Luciferase Assay System 10-Pack from Promega, Lot no. 0000306966
- Passive Lysis Buffer 5x from Promega, Lot No. 0000254353

FURTHER REAGENTS:
- Thiazolyl Blue Tetrazolium Bromide (MTT) solution from Sigma, Lot nos. MKCD4805 and MKCD8033, 5 mg/mL stock solution in deionised Dulbecco’s Phosphate Buffered Saline (DPBS)

CELL CULTURE CONDITIONS: MEDIA
All media were based on Dulbecco’s Modified Eagle Medium (DMEM, Sigma), Lot no. RNBG6681. Depending on the application, the media were supplemented with the following additives:
- Maintainance medium: 9% (v/v) fetal bovine serum ((FBS), Sigma), 1% = geneticin ((G418, Roche, 500 µg/mL)
- Thawing medium: 9.1% FBS
- Test item exposure medium: 1% FBS

CELL CULTURE CONDITIONS: CULTIVATION
- Temperature (°C): 37 ± 1
- CO2 (%): 5

NUMBER OF REPLICATIONS:
Luciferase measurements: triplicates in 3 independent experiments
Cell viability measurement: single measurement in 3 independent runs

EXPERIMENTAL PROCEDURE:
Cells were seeded 10,000 cells / well in a 96-well format and grown for 24 ± 0.5 h in thawing medium. Thereafter, the medium was replaced by 150 µL test item exposure medium and 50 µL of the 4x master concentration.

EXPOSURE: 48 ± 1 h at 37 ± 1 °C

LUCIFERASE ACTIVITY:
After 48 h of exposure the cells were washed 1x with DPBS and 20 µL of lysis buffer were added. Cells were incubated for 20 minutes at room temperature. The plates were transferred into a luminometer where 50 µL of luciferase substrate was added per well. Luciferase activity of each well was assessed for 2 s.

DETERMINATION OF CYTOTOXICITY
After 48 h of test substance exposure, 200 µL of MTT working solution were added to each well, followed by a 4 h incubation period at 37 ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 50 µL of isopropanol. The plate was shaken for 30 minutes and absorption was determined at 570 nm with a spectrophotometer (VarioskanTM LUX Type 3020).

Positive control results:

trans-Cinnamic aldehyde was tested as positive control in a concentration range of 4-64 µM. Cell viability was in the range of the required acceptability criterion of > 70% at all test item concentrations in the first and the third experiment, but below 70% at 32 and 64 µM in the second experiment. Luciferase activity increased dose-dependently and reached an > 1.5-fold induction at a concentration of ≥ 8 µM (first experiment), ≥ 16 µM (second experiment) and ≥ 16 µM (third experiment). The result fits well to the required acceptability criterion of > 1.5-fold induction in at least one of the tested concentrations.

Key result

Run / experiment:

other: 48 h exposure / Experiment 1

Parameter:

other: maximum luciferase activity induction

Value:

12.83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.50 3.6 µg/mL

Key result

Run / experiment:

other: 48 h exposure / Experiment 2

Parameter:

other: maximum luciferase activity induction

Value:

17.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.50: 2.2 µg/mL

Key result

Run / experiment:

other: 48 h exposure / Experiment 3

Parameter:

other: maximum luciferase activity induction

Value:

4.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.50: 1.4 µg/mL

Other effects / acceptance of results:

OTHER EFFECTS:
For the test chemical and positive control substance, in order to derive a prediction, four independent tests were conducted. The results of the first two tests were not concordant and the third test did not meet the acceptance criteria, therefore the third test needed to be repeated. Only the results of three valid tests were reported in the study report.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the assay was demonstrated by the testing facility using the 10 proficiency chemicals listed in the OECD guideline 442D. Moreover, a historical database of data generated with the positive control was maintained over time to confirm the reproducibility of the test method in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control:
1. Yes, the luciferase activity induction of the positive control was > 1.5 in at least one of the tested concentrations and statistically significant compared to the solvent (DMSO) control.
2. No, the average induction (Imax) in the 3 replicates for the positive control at a concentration of 64 µM was not within the historical range (2-8 -fold) but 28.35, 83.88 and 2.45-fold in the last repeated test. Because a clear dose-response relationship was observed for luciferase induction, the test was nevertheless considered valid.
3. Yes, the EC 1.5 value of the positive control was 6 µM, 9 µM and 14 µM and fell within two standard deviations of the historical mean (5.6-23.7 µM).
- Acceptance criteria met for variability between replicate measurements: Yes, the average coefficient of variation (CV) of the luciferase activity for the negative (DMSO)/blank control was < 20% in each repetition (18.17%, 18.86% and 16.86% respectively).

Table 1: Summary of results with the test item

Number of tests	Significant induction above 1.5-fold	Viability > 70 % at lowest concentration with ≥ 1.5 fold	EC1.5 < 1000 µM or 200 µg/ml	Showing clear dose response (yes/no)	KeratinoSens™ result obtained
	(yes/no)				(positive/negative/inconclusive-repeat)
1	yes	no	yes	yes	negative
2	yes	yes	yes	yes	positive
3	yes	yes	yes	yes	positive
- repeated
2 out of 3 approach					positive

Table 2: Experimental results of the test item

	1. Experiment			2. Experiment			3. Experiment
Concentration (uM)	average induction mean of 3 replicates	significance	viability	average induction mean of 3 replicates	significance	viability	Concentration (uM)	average induction mean of 3 replicates	significance	viability
1	1.08	0.308	82%	1.24	0.231	235%	0.7	1.64	0.014	82%
2	1.03	0.801	127%	1.25	0.321	102%	1.1	1.09	0.687	95%
4	1.1	0.434	108%	1.09	0.643	86%	1.6	1.25	0.041	85%
8	1.17	0.085	109%	1.62	0.075	85%	2.4	1.23	0.066	88%
16	1.43	0.028	81%	1.78	0.042	93%	3.7	1.18	0.261	95%
31	1.87	0.001	64%	2.41	0	85%	5.5	1.27	0.231	102%
63	3.88	0.018	33%	3.32	0.05	23%	8.2	1.64	0.001	102%
125	12.83	0.088	1%	17.7	0.024	3%	12.3	1.65	0.027	108%
250	0	0	0%	-0.02	0.001	0%	18.5	1.72	0.002	120%
500	-0.01	0	1%	-0.04	0.001	1%	27.8	1.63	0.024	125%
1000	-0.02	0	4%	-0.05	0.001	3%	41.7	2.91	0	115%
2000	-0.02	0	13%	-0.05	0.001	21%	62.5	4.76	0.016	107%

Table 3: Experimental results of the positive control

	Concentration (µM)	average induction	significance	viability
1. Experiment	4	1.32	0.064	100%
	8	1.66	0.004	90%
	16	1.64	0.002	92%
	32	2.61	0	94%
	64	28.35	0.022	75%
2. Experiment	4	1.11	0.567	92%
	8	1.38	0.103	95%
	16	2.27	0.022	91%
	32	3.38	0.002	45%
	64	83.88	0.014	5%
3. Experiment	4	1.21	0.144	107%
	8	1.24	0.336	109%
	16	1.58	0.049	113%
	32	1.92	0.019	125%
	64	2.45	0.005	141%

Interpretation of results:

other: positive for kerationcyte activation

Conclusions:

The data generated with this method may not be sufficient to conclude on the absence of a skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Two in vitro studies on skin sensitisation are available for test substance evaluation, comprising a test on dendritic cell activation as well as an assay on the activation of keratinocytes.

 

Activation of dendritic cells: h-CLAT test

The test item was analysed for its skin sensitising potential using the human Cell Line Activation Test (hCLAT) according to OECD guideline 442E and in compliance with GLP (Katona, 2019a). The test quantifies changes in the expression of the cell surface marker proteins CD54 and CD86 on dendritic cells in response to sensitisers. The human leukemia cell line THP-1 was used for the test.

In a first step, two independent dose-finding assays were performed to identify the mean concentration of the test item that results in 75% cell viability (CV75) compared to the solvent control. Based on the CV75 value, a concentration of 1.2 x CV75 was chosen as highest concentration for the hCLAT test. THP-1 cells were exposed for 24 ± 0.5 h at 37 °C to eight different test item concentrations in the range of 15 to 54 µg/mL. In addition, vehicle (Dimethylsulfoxide, (DMSO)) and positive controls (1-chloro-2,4-dinitrobenzene (DNCB)) were included. Each concentration was tested in triplicates. After 24 h of exposure, cells were stained with fluorescein isothiocyanate labelled antibodies against the cell surface proteins CD54 and CD86 and the expression of CD54 and CD86 was quantified by flow cytometry.

The results of the positive and the negative controls demonstrated the validity of the test. The test item showed an increased CD86 marker expression (relative fluorescence intensity (RFI) ≥ 150 % in two out of three experiments and an increased CD54 marker expression (RFI ≥ 200 %) in all three experimental runs. The result is indicative for dendritic cell activation.

 

Inflammatory response in keratinocytes: KeratinoSens^TM assay

A KeratinoSens^TM assay was performed in accordance with OECD 442D and in compliance with GLP (Katona, 2019b) to assess the keratinocyte activating potential of the test item. The test uses the transgenic KeratinoSens cell line HaCaT, which stably expresses the antioxidant/electrophilic response element (ARE)- dependent luciferase reporter gene under the control of the ARE-element and thus enables the indication of cyto-protective signaling pathways in response to electrophiles and oxidative stress.

The test item, as well as negative and positive controls were incubated with the transgenic keratinocytes for 48 ± 1 h at 37 ± 1 °C, followed by determination of luciferase activity and assessment of cytotoxicity. Twelve concentrations of the test substance in the range of 1 -2000 µM were included, as well as a concentration range of 4-64 µM of the positive control trans-cinnamaldehyde. DMSO (1% in cultivation medium) served as solvent control. Three independent experiments were performed, using each three replicates per condition.

After 48 h of exposure, luciferase activity was measured and cytotoxicity was determined using the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. Results were only considered to be reliable if the cell viability was > 70%. A dose-dependent, statistically significant luciferase induction > 1.5-fold was achieved with the positive control substance, while the luminescence reading of the negative control showed no luciferase induction (coefficient of variation < 20%), confirming the validity of the test system.

The maximal luciferase activity induction of the test item was > 1.5-fold in the first (at concentrations ≥ 31 µM) and in the second experiment (at concentrations ≥ 8 µM). However, the cell viability in the first experiment was < 70% at the lowest concentration with 1.5-fold induction. Thus, a third experiment was performed with test item concentrations in the range of 0.7-62.5 µM. The experiment fulfilled all acceptance criteria and revealed a significant > 1.5-fold induction of luciferase activity at test item concentrations of ≥ 8.2 µM, confirming the positive test result obtained in the previous experiments.

In summary, the test item induced luciferase activity in two out of three experiments.

 

Conclusion

Two in vitro studies on skin sensitisation are available, one on the activation of dendritic cells, one on the activation of keratinocytes. Both in vitro tests revealed a positive result. As two out of three possible in vitro/in chemico tests are positive for skin sensitisation, the test item is classified based on a weight of evidence approach as Skin Sens. 1 (H317) according to Regulation (EC) No. 1272/2008 (CLP).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification as Skin Sens. Cat. 1 (H317) according to Regulation (EC) No. 1272/2008.