Reaction mass of 1,5-naphthylene diisocyanate and Amines, hydrogenated tallow alkyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 2018 to 02 March 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:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: KeratinoSens Assay

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Molecular formula:

C24H32N4O2

IUPAC Name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Test material form:

solid

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell Culture;
Basic medium; Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Maintenance medium; Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
Exposure medium; Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

In the main experiments the test item was suspended in dimethyl sulfoxide (DMSO) at 50 mM. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. In the first experiment precipitation was observed at the start of the incubation period at concentrations of 31 µM and upwards and at the end of the incubation period at concentrations of 125 µM and upwards. In the second experiment precipitation was observed at the start and end of the incubation period at concentrations of 125 µM and upwards.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 – 98 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8 – 36.4 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Experimental Design
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+12 in experiment 1 and P+7 in experiment 2.

For the treatment of cells, the medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.

To measure the Luciferase Activity, the Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

For the cytotoxicity assessment, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Results and discussion

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.89 and the EC1.5 22 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.41 and the EC1.5 73 µM.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: other: Experiment 1

Parameter:

other: Imax [442D] (migrated information)

Value:

1.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Precipitation was observed at the start of the incubation period at concentrations of 31 µM and upwards and at the end of the incubation period at concentrations of 125 µM and upwards

Results 2

Run / experiment:

other: other: Experiment 2

Parameter:

other: Imax [442D] (migrated information)

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Precipitation was observed at the start and end of the incubation period at concentrations of 125 µM and upwards.

Results 3

Run / experiment:

other: other: Experiment 1 and 2

Parameter:

other: IC30 [442D] (migrated information)

Value:

0.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test item precipitated at dose levels of 125 µM and upwards in experiment 1 and 2, respectively.

Results 4

Run / experiment:

other: other: Experiments 1 and 2

Parameter:

other: IC50 [442D] (migrated information)

Value:

0.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The test item precipitated at dose levels of 125 µM and upwards in experiment 1 and 2, respectively.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test item showed toxicity (IC30 and IC50 values of < 0.24 µM in both experiments). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.11-fold and 1.12-fold in experiment 1 and 2 respectively. The test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (11 % and 8.0 % in experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 µM (22 µM and 73 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.89- and 2.41-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (11% and 8.0 6% in experiment 1 and 2, respectively).

Any other information on results incl. tables

Table 1: Overview Luminescence Induction and Cell Viability of Reaction mass of aniline and m-tolylidene diisocyanate in Experiment 1 and 2

Concentration (µM)	0.24	0.49	0.98	2.0	3.9	7.8	16	31	63	125	250	500
Exp. 1 luminescence	0.98	0.85	0.92	1.01	1.05	1.03	1.03	1.07	1.06	1.11	1.00	1.02
Exp. 1 viability (%)	21.9	24.2	27.4	25.4	29.4	27.9	27.9	29.3	34.3	24.9	22.7	30.0
Exp. 2 luminescence	0.88	0.93	1.00	1.01	1.04	1.04	1.09	1.12	1.07	1.06	1.12	1.10
Exp. 2 viability (%)	34.0	37.6	34.9	36.6	34.2	36.3	40.6	39.4	36.7	35.2	35.3	34.4

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp. 1 Luminesence	1.33	1.40	1.65***	1.85***	2.07***	2.89***
Exp. 1 viability (%)	96.7	95.9	104.8	102.1	105.2	107.5
Exp. 2 Luminesence	0.99	1.13	1.25	1.44	1.79***	2.41***
Exp. 2 viability (%)	114.6	107.7	121.6	126.4	133.3	130.3

*** p <0.001 Student's t-test

Table 3: Overview EC_1.5, I_max, IC₃₀ and IC₅₀ Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test Item; Experiment 1	NA	1.11	NA	NA
Test Item; Experiment 2	NA	1.12	NA	NA
Pos. Control; Experiment 1	22	2.89	NA	NA
Pos. Control; Experiment 2	73	2.41	NA	NA

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item showed toxicity in a KeratinoSens assay (IC30 values and IC50 values of < 0.24 µM in both experiments). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.11-fold and 1.12-fold in experiment 1 and 2 respectively. The test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM.

Executive summary:

The skin sensitisation of the test item was assessed in a KerationSens Assay with procedure based on the OECD TG 442D guidance, in two independent experiments in triplicate. The test item was suspended in the vehicle solvent dimethyl sulfoxide (DMSO) prior to dilution with test media and the experiment was performed at final test concentrations of 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 µM (final concentration DMSO of 1%).  In the first experiment precipitation was observed at the start of the incubation period at concentrations of 31 µM and upwards and at the end of the incubation period at concentrations of 125 µM and upwards. In the second experiment precipitation was observed at the start and end of the incubation period at concentrations of 125 µM and upwards. Alongside the test item a solvent (negative control) of DMSO and positive control of Ethylene dimethacrylate glycol (EDMG), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%).

The test item showed toxicity in a KeratinoSens assay (IC30 values and IC50 values of < 0.24 µM in both experiments).  No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 1.11-fold and 1.12-fold in experiment 1 and 2 respectively.  The test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM.