reaction product of toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate) and aniline (molarratio 1:2)

Endpoint summary

Description of key information

The skin sensitisation potential of one TDI-II category member, Reaction mass of aniline and m-tolylidene diisocyanate, was assessed. The results from the KeratinoSens^TM assay were inconclusive for sensitisation potential. No induction of luciferase activity was observed in the first experiment. However, induction was observed in the second experiment (EC 1.5 value of 57 µg/mL), but this was only observed at a concentration with a cell viability <70%, and therefore this induction is not considered biologically relevant. Negative results were observed at test concentrations up to 100 µg/mL (Westerink 2018).

In the U-Sens^TM assay precipitation was observed at concentrations below 200 µg/mL, which means the negative results observed are not considered conclusive. The U-Sens™ assay was therefore concluded to be positive according to the requirements of OECD Test Guideline 442E (Eurlings 2018).

 

A DPRA assay could not be performed on the substances in this category based on the applicability domain. Therefore, no conclusion on skin sensitisation properties and potency can be drawn from the in vitro tests performed and an LLNA assay according to the OECD 429 guideline was conducted.

In the LLNA assay conducted according to OECD 429 guideline, mean DPM/animal values for the experimental groups treated with test item concentrations of 2, 5 and 10% were 1158, 1110 and 1260 DPM, respectively. The mean DPM/animal value for the vehicle control group was 896 DPM. The SI values calculated for the test item concentrations of 2, 5 and 10% were 1.3, 1.2 and 1.4, respectively. Variation in ear thickness during the observation period exceeded 25% for four animals treated at 5% and four animals treated at 10%. As no clear correlation was seen with the DPM values and the outcome of the study was negative, these findings were considered not to have affected the results of the study (van Sas 2018). Based on these results, the test item would not be regarded as a skin sensitiser according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21/03/2018 - 30/04/18

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)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

other: EC No. 640/2012 Part B (Skin Sensitization: Local Lymph Node Assay) July 2012

GLP compliance:

yes (incl. QA statement)

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: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old
- Weight at study initiation: 19.0 - 24.4 g
- Housing: Group housed in groups of 5
- Diet (e.g. ad libitum): Pelleted rodent diet
- Water (e.g. ad libitum): Tap water freely available
- Acclimation period: 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 41-51
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light): 12 hour light: 12 hour dark

Vehicle:

dimethyl sulphoxide

Concentration:

0, 2, 5, 10% w/w

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 10%
- Irritation: Slight erythema
- Systemic toxicity: No signs
- Ear thickness measurements: Variation - 25%

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: SI > 3

TREATMENT PREPARATION AND ADMINISTRATION: Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing.
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day.

Positive control substance(s):

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

Statistics:

Stimulation index (SI) calculated for each group using individual SI values

Positive control results:

An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was found to be within the acceptable range of 4.8 and 19.5%.

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

Control

Remarks on result:

other: Mean SI group value used

Parameter:

SI

Value:

1.3

Variability:

0.1

Test group / Remarks:

2%

Remarks on result:

other: Mean SI group value used

Parameter:

SI

Value:

1.2

Variability:

0.2

Test group / Remarks:

5%

Remarks on result:

other: Mean SI group value used

Parameter:

SI

Value:

1.4

Variability:

0.1

Test group / Remarks:

10%

Remarks on result:

other: Mean SI group value used

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION : Individual SI is the ratio of the DPM/animal compared to the DPM/control group mean

CLINICAL OBSERVATIONS: No clinical signs of systemic toxicity

BODY WEIGHTS: Body weights remained in same range

group	TS^1 (%)	animal	Size nodes^2		DPM^3/ animal	mean DPM ± SEM^4			mean SI ± SEM
			left	right
1	0	1	n	n	802	896	±	117	1.0	±	0.1
		2	n	n	717
		3	n	n	1151
		4	n	n	1193
		5	n	n	617
2	2	6	n	n	1232	1158	±	132	1.3	±	0.1
		7	n	n	984
		8	n	n	797
		9	n	n	1586
		10	n	n	1191
3	5	11	n	n	467	1110	±	197	1.2	±	0.2
		12	n	n	1403
		13	n	n	922
		14	n	n	1603
		15	n	n	1154
4	10	16	n	n	1256	1260	±	129	1.4	±	0.1
		17	n	n	1316
		18	n	n	1708
		19	n	n	1068
		20	n	n	954

Interpretation of results:

GHS criteria not met

Conclusions:

Test item was determined not to be a skin sensitiser.

Executive summary:

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 and 10% on three consecutive days by open application to the ears. Five vehicle control mice were also treated with the vehicle alone (dimethyl sulfoxide). Three days after the last exposure, all animals were injected with the ³H-methyl thymidine and after five hours the draining lymph nodes were excised and pooled for each animal. After precipitating the DNA of lymph node cells, radioactivity measurements were performed. The SI values calculated for the test item at concentrations of 2, 5 and 10% were 1.3, 1.2 and 1.4 respectively. There was no indication that the test item elicited a SI≥3 when tested up to 10%. Therefore, the test item was determined not to be a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2017 to 19 January 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)

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).

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 dissolved in dimethyl sulfoxide (DMSO) at 10 mg/ml. 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 100, 50, 25, 13, 6, 3, 2, 1, 0.4, 0.2, 0.1 and 0.05 µg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. However, precipitation was observed at the start and end of the incubation period at the top concentration of 100 µg/mL in the 96-well plates.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 – 36.6°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±1.0oC 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.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.17 and the EC1.5 76 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.16 and the EC1.5 90 µM.

Run / experiment:

other: other: Experiment 1

Parameter:

other: IC30 (ug/L)

Value:

0.51

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Run / experiment:

other: other: Experiment 2

Parameter:

other: IC50 (µg/mL)

Value:

0.64

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Run / experiment:

other: other: Experiment 1

Parameter:

other: Imax (µg/mL)

Value:

1.23

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Run / experiment:

other: other: Experiment 2

Parameter:

other: IC30 (µg/mL)

Value:

0.38

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Run / experiment:

other: other: Experiment 2

Parameter:

other: IC50 (µg/mL)

Value:

0.55

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Run / experiment:

other: other: Experiment 2

Parameter:

other: Imax (µg/mL)

Value:

1.94

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Run / experiment:

other: other: Experiment 2

Parameter:

other: EC1.5 (µg/mL)

Value:

57

Vehicle controls validity:

not specified

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 the top concentration of 100 µg/mL in the 96-well plates

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test item showed toxicity (IC30 values of 0.51 µg/mL and 0.38 µg/mL and IC50 values of 0.64 µg/mL and 0.55 µg/mL in experiment 1 and 2, respectively). No induction of the luciferase activity was observed in the first experiment. An induction of the luciferase activity (EC1.5 value of 57 µg/mL) was measured in the second experiment. However, the luciferase induction was only observed at a concentration with a cell viability <70%. Therefore, this induction was not considered biologically relevant. The maximum luciferase activity induction (Imax) was 1.23-fold and 1.94-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 up to test concentrations of 100 µg/mL.

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% (8.3% and 10.6% 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 (76 µM and 90 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.16-fold in both experiments).
- 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% (8.3% and 10.6% in experiment 1 and 2, respectively).
- Range of historical values if different from the ones specified in the test guideline:

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

Concentration (µg/mL)	0.049	0.10	0.20	0.39	0.78	1.6	3.1	6.3	13	25	50	100
Exp. 1 luminescence	0.89	0.86	0.91	1.00	1.019	1.23	1.19	1.06	1.11	1.10	1.02	1.13
Exp. 1 viability (%)	110.4	97.6	92.7	88.1	27.9	41.9	28.9	26.1	23.3	24.1	23.5	26.1
Exp. 2 luminescence	1.06	1.02	1.11	1.17	1.29	0.34	1.46	1.16	1.25	1.38	1.43	1.94
Exp. 2 viability (%)	88.7	83.1	84.8	69.3	22.8	1.4	28.4	18.5	18.4	20.7	25.0	25.1

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	1.03	1.05	1.15	1.33	2.17	2.16
Exp. 1 viability (%)	99.8	99.9	108.6	116.0	118.3	132.1
Exp. 2 Luminesence	0.89	1.01	1.17	1.29	1.78	2.16
Exp. 2 viability (%)	88.8	89.4	85.0	92.6	94.5	99.3

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

	Imax	IC30	IC50
Test Item; Experiment 1	1.23 µg/mL	0.81 µg/mL	0.64 µg/mL
Test Item; Experiment 2	1.94 µg/mL	0.38 µg/mL	0.55 µg/mL
Pos. Control; Experiment 1	2.17 µM	NA	NA
Pos. Control; Experiment 2	2.16 µM	NA	NA

NA = Not applicable

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item showed toxicity in a KeratinoSens assay (IC30 values of 0.51 µg/mL and 0.38 µg/mL and IC50 values of 0.64 µg/mL and 0.55 µg/mL in experiment 1 and 2, respectively). No induction of the luciferase activity was observed in the first experiment. An induction of the luciferase activity (EC1.5 value of 57 µg/mL) was measured in the second experiment. However, the luciferase induction was only observed at a concentration with a cell viability <70%. Therefore, this induction was not considered biologically relevant. The maximum luciferase activity induction (Imax) was 1.23-fold and 1.94-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 up to test concentrations of 100 µg/mL.

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 100, 50, 25, 13, 6, 3, 2, 1, 0.4, 0.2, 0.1 and 0.05 µg/mL (final concentration DMSO of 1%). All formulations formed a clear solution, however, precipitation was observed at the start and end of the incubation period at the top concentration of 100 µg/mL in the 96-well plates. 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 (IC₃₀values of 0.51µg/mLand 0.38µg/mLand IC₅₀values of 0.64µg/mL and 0.55 µg/mLin experiment 1 and 2, respectively). No induction of the luciferase activity was observed in the first experiment. An induction of the luciferase activity (EC_1.5value of 57µg/mL) was measured in the second experiment. However, the luciferase induction was only observed at a concentration with a cell viability <70%. Therefore, this induction was not considered biologically relevant. The maximum luciferase activity induction (I_max) was 1.23-fold and 1.94-fold in experiment 1 and 2 respectively. The test itemis classified as inconclusive in the KeratinoSens assay since negative results (<1.5-fold induction) were observed up to test concentrations of 100 µg/mL.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 December 2017 to 5 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: U937 Cell Line Activation Test

Justification for non-LLNA method:

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

Details on the study design:

U937 human monocytes (ATCC (American Type Culture Collection, Virginia, USA). ATCC no.: CRL-1593.2TM) were used in the study as they are inducible CD86-expressing cells. Stock cultures of these cells are stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 180, 140 and 100 µg/mL in the 96-well plate in experiment 1 and 2, respectively (final concentration DMSO of 0.4%). Test item concentrations were used within 3.5 hours after preparation and any residual volumes were discarded.

All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 90 – 101 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 – 36.6°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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. If the cell viability is < 90% the cells were not used. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of complete medium untreated control, solvent/vehicle control, negative and positive controls were tested. At least two experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. In total 2 valid experiments were performed.

Cells are treated for 45 ± 3 hours with the selected doses. The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL. A negative untreated control (culture medium), a vehicle control and the positive and negative control items were included. The final volume in the wells was 200 µL. In the second experiment and following, if applicable, cells were treated with four selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 100, 140, 180 and 200 µg/mL. After 45 ± 3 hours of exposure, wells were checked for precipitate.

Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells. FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

A flow cytometry method was used for analysis. Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. When the cell viability was low, up to 20,000 cells including dead cells could be acquired. Alternatively, data can be acquired for one minute after the initiation of the analysis. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.

All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
The results were interpreted as follows:

- For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
- The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 Geo Mean S.I. ≥ 150%).
- In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
- An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
- An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.

The exception is if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration - see paragraph 27) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently) (Figure 1). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).

Positive control results:

In Experiment 1: The positive control (TNBS) showed a S.I. ≥ 296% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. < 111% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
In Experiment 2: The positive control (TNBS) showed a S.I. ≥ 533% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. < 121% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Run / experiment:

other: other: Experiment 1

Parameter:

other: EC150 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: other: Experiment 1

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: other: Experiment 2

Parameter:

other: EC150 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: other: Experiment 2

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test item showed no toxicity (CV70 value >200 µg/mL) and no biologically relevant induction of the CD86 activity (EC150 value >200 µg/mL) was measured at any of the test concentrations in both experiments. However, the test item precipitated at the dose levels of 50, 100 and 200 µg/mL and 100, 140, 180 and 200 µg/mL in experiment 1 and 2, respectively.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Acceptance criteria were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control (LA) showed a S.I. < 111 % and < 121 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%), in Experiments 1 and 2, respectively, and the acceptance criteria were met.
- Acceptance criteria met for positive control: The positive control (TNBS) showed a S.I. ≥ 296 % and ≥ 533 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%) in Experiments 1 and 2, respectively and therefore the acceptance criteria were met.
- Acceptance criteria met for variability between replicate measurements: At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (96% in experiment 1 and 2). The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (97% in experiment 1 and 98% in experiment 2).

• The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments.
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and
≤ 25% in both experiments.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of
≥ 0.6% and < 1.5% in both experiments.
• No drift in CD86 expression was observed in the untreated controls and negative controls.

Table 1: Overview of Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of Reaction mass of aniline and m-tolylidene diisocyanate

Test Item	Dose (µg/mL)	% Viability Mean		CD86 -IgG1 S.I.		Colour Interference S.I.
		Experiment		Experiment		Experiment
		1	2	1	2	1	2
Reaction mass of aniline and m-tolylidene diisocyanate	1	97	-	110	-	98	-
	10	97	-	92	-	97	-
	20	97	-	94	-	92	-
	50	97	-	100	-	92	-
	100	96	98	113	84	90	81
	140	-	98	-	41	-	99
	180	-	98	-	36	-	98
	200	96	99	109	95	90	85

- Not applicable

Table 2: Overview of Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls	% Viability (Mean)		CD86 -IgG1 S.I.
	Experiment		Experiment
	1	2	1	2
LA1	96	96	111	100
LA2	96	96	111	121
LA3	96	96	106	97
TNBS1	92	90	477*	533*
TNBS2	93	90	452*	533*
TNBS3	92	89	296*	618*
DMSO	97	98	111	178
	IG1 value (%)		CD86 basal expression (%)
	Experiment		Experiment
	1	2	1	2
RPMI1	0.6	7.8	0.7	4.1
RPMI2	1.1	8.7	1.4	4.4
RPMI3	0.8	8.7	0.5	4.0
RPMI Mean Variability			96	96
RPMI Drift			7 %	9 %
LA Drift			-5 %	-12 %

* Result either below 70 % viability or above 150 S.I..

Table 3: Overview of EC₁₅₀ and CV₇₀ values

	EC150 (µg/mL)	CV70 (µg/mL)
Test item Experiment 1	NA	NA
Test item Experiment 2	NA	NA

NA = Not applicable

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item showed no toxicity (CV70 value >200 µg/mL) and no biologically relevant induction of the CD86 activity (EC150 value >200 µg/mL) was measured at any of the test concentrations in both experiments in a U-Sens™ assay. The test item is, however, classified as Positive in the U-Sens™ assay since precipitation is observed in both experiments.

Executive summary:

The skin sensitisation potential of the test item was assessed in a U-Sens assay designed to quantify the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The experiment was designed based on OECD 442E – Annex II ‘In Vitro Skin Sensitisation:  U937 Cell Line Activation Test (U-SENS™)’ (9 October 2017).

 

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 180, 140 and 100 µg/mL in the 96-well plate in experiment 1 and 2, respectively. In both experiments concurrent solvent, negative and positive controls of DMSO, Lactic acid and 2,4,6-Trinitrobenzenesulfonic acid (TNBS) were also investigated.

 

The test item showed no toxicity (CV70 value >200 µg/mL) and no biologically relevant induction of the CD86 activity (EC150 value >200 µg/mL) was measured at any of the test concentrations in both experiments in a U-Sens™ assay and the acceptance criteria were met for the positive, negative and solvent control. The test item is, however, classified as Positive in the U-Sens™ assay since precipitation is observed in both experiments, with precipitation observed at the end of the incubation period at 50, 100 and 200 µg/mL in experiment 1 and at 100, 140, 180 and 200 µg/mL in experiment 2.

 

The study is a GLP compliant guideline experimental study, and is acceptable without restrictions for the assessment of this endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The KeratinoSens^TM assay was inconclusive based on negative results at concentrations with sufficient cell viability up to 100 µg/mL, and luciferase induction in one experiment only at cytotoxic concentrations where cell viability was below 70%, making the biological relevance of induction questionable. In the U-Sens^TM assay, precipitation was observed at concentrations below 200 µg/mL, which means the negative results at these concentrations are not considered conclusive. The U-Sens™ assay was therefore concluded to be positive according to OECD 442E.

In the LLNA assay conducted according to the OECD 429 guideline, mean DPM/animal values for the experimental groups treated with test item concentrations of 2, 5 and 10% were 1158, 1110 and 1260 DPM, respectively. The mean DPM/animal value for the vehicle control group was 896 DPM. The SI values calculated for the test item concentrations of 2, 5 and 10% were 1.3, 1.2 and 1.4, respectively. Variation in ear thickness during the observation period exceeded 25% for four animals treated at 5% and four animals treated at 10%. As no clear correlation was seen with the DPM values and the outcome of the study was negative, these findings were considered not to have affected the results of the study. Based on these results, the test item would not be regarded as a skin sensitiser according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.

Justification for classification or non-classification

Based on the available in vitro data available for Reaction mass of aniline and m-tolylidene diisocyanate, no assessment of classification can be made, as the results were inconclusive. Therefore, in accordance with the signed study plan, an LLNA was performed to assess the classification requirement for TDI category II substances. In the LLNA assay conducted according to the OECD 429 guideline, mean DPM/animal values for the experimental groups treated with test item concentrations of 2, 5 and 10% were 1158, 1110 and 1260 DPM, respectively. The mean DPM/animal value for the vehicle control group was 896 DPM. The SI values calculated for the test item concentrations of 2, 5 and 10% were 1.3, 1.2 and 1.4, respectively. Variation in ear thickness during the observation period exceeded 25% for four animals treated at 5% and four animals treated at 10%. As no clear correlation was seen with the DPM values and the outcome of the study was negative, these findings were considered not to have affected the results of the study. Based on these results, the test item would not be regarded as a skin sensitiser according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact. This conclusion is considered to be appropriate for all TDI-II category substances.