3,4-dihydroxybenzonitrile

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 November - 07 December 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

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. DPRA is one of the recommended in chemico test systems in the international OECD guidelines.

Details on the study design:

Principle of the assay
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration was measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine- and lysine peptide Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Test system
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL.
Source and batch
JPT Peptide Technologies GmbH, Berlin, Germany.
Batch No.280916K-44
Storage: The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.

Positive control
Cinnamic aldehyde, kosher (104-55-2)

Reagents
Acetonitrile (ACN) HPLC-grade, Fisher Chemicals, Loughborough, England
Ammonium acetate (CH3COONH4) Fractopur, Biosolve, Valkenswaard, The Netherlands
Ammonium hydroxide (NH4OH) 25.0% Merck, Darmstadt, Germany
Disodiumhydrogenphosphate Emsure, Merck
(Na2HPO4.12H2O)
Milli-Q water (MQ) Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion-exchange cartridges Sodiumdihydrogenphosphate Emsure, Merck
(NaH2PO4.H2O)
Trifluoroacetic acid, >99% (TFA) Sigma Aldrich, Zwijndrecht, The Netherlands

Test item preparation
CH02906 stock solutions were prepared freshly for each reactivity assay. For the cysteine and lysine reactivity assay respectively 19.78 mg and 13.49 mg of CH02906 were pre-weighed into a clean amber glass vial and dissolved, just before use, in respectively 1464 µL and 998 µL acetonitryl (ACN) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

HPLC-PDA analysis
The concentration of SPCC or SPCL in the samples was measured by HPLC-PDA. Sample analysis was performed using the following system:
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)

Cysteine reactivity assay
The reactivity of CH02906 towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis following 24±2 hours of incubation at 25±2.5°C.

Lysine reactivity assay
The reactivity of CH02906 towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis following 24±2 hours of incubation at 25±2.5°C.

The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variability (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Run / experiment:

other: Cysteine

Parameter:

other: mean SPCC A220/A258 area ratio

Value:

14.03

Positive controls validity:

valid

Key result

Run / experiment:

other: Cysteine

Parameter:

other: mean Percent SPCC Depletion

Remarks:

%

Value:

13.7

Remarks on result:

no indication of skin sensitisation

Remarks:

SD ± 5.1%. the Cysteine 1:10 prediction model was used for classification due to co-elution of CH02906 with SPCL

Run / experiment:

other: lysine

Parameter:

other: mean SPCL A220/A258

Remarks on result:

not determinable

Remarks:

the mean SPCL A220/A258 could not be calculated since co-elution of CH02906 with SPCL occurred

Run / experiment:

other: lysine

Parameter:

other: Percent SPCL Depletion

Remarks on result:

not determinable

Remarks:

As a result of co-elution, the Percent SPCL Depletion could not be calculated for CH02906

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
Acceptability of the cysteine reactivity assay
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.992. Since the r2 was >0.990, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.498±0.011 mM. The mean peptide concentration of Reference Controls C was 0.526±0.011 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve CH02906 did not impact the Percent SPCC Depletion.
The SPCC peak areas for Reference controls B and C are presented in Table 9. The CV of the peptide areas for the nine Reference Controls B and C was 2.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 14.55. The mean A220/A258 ratio ± 10% range was 13.10 - 16.01. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.7% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the lysine reactivity assay
The mean peptide concentration of Reference Controls A was 0.470±0.017 mM. The mean peptide concentration of Reference Controls C was 0.465±0.015 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve CH02906 did not impact the Percent SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 3.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 14.81. The mean A220/A258 ratio ± 10% range was 13.33 - 16.29. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The results of the positive control cinnamic aldehyde are presented in Table 16. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 44.6% ± 3.1%. This was within the acceptance range of 40.2 - 69%, with a SD that was below the maximum criteria (SD <11.6%).

Interpretation of results:

GHS criteria not met

Conclusions:

It can be concluded that this DPRA test is valid, and that CH02906 was negative in the DPRA, and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.

Executive summary:

In this study the reactivity of CH02906 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined. Following 24 hours of incubation of CH02906 with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The study procedures described in this report were based on the most recent OECD guidelines.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve CH02906, and was therefore used in this DPRA study.

The correlation coefficient (r²) of the SPCC and SPCL standard calibration curves were 0.992 and 0.996, respectively. The calibration curves were within the acceptance criteria, since the r²for both curves was >0.990.

For the cysteine reactivity assay, the mean peptide concentration of Reference Controls A was 0.498±0.011mM while the mean peptide concentration of Reference Controls C was 0.526±0.011mM. The means of Reference Control samples A and C were within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system, and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The Coefficient of Variability (CV) of peptide areas for the nine Reference Controls B and C was 2.9%. This was within the acceptance criteria (CV<15.0%), and confirms the stability of the HPLC run over time. The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 14.55. The mean A₂₂₀/A₂₅₈ratio± 10% range was13.10-16.01. Each sample showing an A₂₂₀/A₂₅₈ratio within this range gives an indication that co-elution has not occurred.

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.7%± 1.0%. This was within the acceptance range of 60.8 to 100% with a standard deviation (SD) that was below the maximum (SD <14.9%).

For CH02906, the mean SPCCA₂₂₀/A₂₅₈area ratio was 14.03. Since this is within the 13.10-16.01 range this indicates that there is no co-elution ofCH02906 with SPCC. The mean Percent SPCC depletion for CH02906 was 13.7%± 5.1%.

For the lysine reactivity assay, the mean peptide concentration of Reference Controls A was             0.470±0.017mM while the mean peptide concentration of Reference Controls C was 0.465±0.015mM. The means of Reference Control samples A and C were within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system, and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The CV of peptide areas for the nine Reference Controls B and C was 3.9%. This was within the acceptance criteria (CV<15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 14.81. The mean A₂₂₀/A₂₅₈ratio± 10% range was13.33 - 16.29. Each sample showing an A₂₂₀/A₂₅₈ratio within this range gives an indication that co-elution has not occurred.

The mean Percent SPCL Depletion for cinnamic aldehyde was 44.6% ± 3.1%. This was within the acceptance range of 40.2 to 69.0%, with a SD that was below the maximum (SD <11.6%).

ForCH02906, the mean SPCL A₂₂₀/A₂₅₈could not be calculated since co-elution ofCH02906with SPCL occurred. As a result of this co-elution, the Percent SPCL Depletion could not be calculated for CH02906.

An overview of the individual results of the cysteine and lysine reactivity assays are presented in Table1. In the cysteine reactivity assay, CH02906 showed 13.7% SPCC depletion while in the lysine reactivity assay co-elution of CH02906 with the lysine peptide occurred. CH02906 was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 prediction model. Therefore, CH02906 was considered to be negative in the DPRA.

Table1: SPCC and SPCL depletion and reactivity classification forCH02906

Test item	SPCC depletion		SPCL depletion		Reactivity class
	Mean	± SD	Mean	± SD	Cysteine 1:10 prediction model
CH02906	13.7%	5.1%	NA, co-elution		No or minimal reactivity

                                   SD = Standard Deviation, NA = Not Applicable.

It can be concluded that this DPRA test was valid and that CH02906 was negative in the DPRA, and is classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 February - 10 March 2017

Reliability:

2 (reliable with restrictions)

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 February, 2015

Deviations:

yes

Remarks:

In the second experiment, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was 20.2%.

GLP compliance:

yes

Type of study:

activation of keratinocytes

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 (OECD 442D).

Details on the study design:

Test System
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 (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell culture
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Maintenance medium: Dulbecco’s minimal 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 supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 37.3°C).

Subculturing
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 after starting-up

Vehicle
Dimethyl Sulfoxide
The solvent control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

Negative Control
The vehicle of the test substance, i.e. dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

Positive Control
Ethylene dimethacrylate glycol (CAS 97-90-5). Ethylene dimethacrylate glycol was used as positive control since Charles River Laboratories Den Bosch has an historical database for this sensitizer. Moreover Ethylene dimethacrylate glycol is like cinnamic acid a weak sensitizer but gave more stable results than cinnamic acid. Laboratory proficiency was shown in Charles River Laboratories Den Bosch project 511093.
The concentration of the positive control ranged from 7.81 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

Dose Formulation
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM. The 100-fold dilution in DMEM of the 200 mM stock showed no precipitation (final concentration 2000 µM). This concentration was selected as highest concentration for the main assay (highest concentration required by the guideline).
The stock and spike solutions were diluted 25-fold with exposure medium (concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 µM (final concentration DMSO of 1%).

Two independent experiments were performed

Experimental Design
Plating of cells: 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 test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator.
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 control substances and the 11 diluted stock solutions were added, resulting in the final test concentrations of the test chemical (4-fold dilution of the formulations in 4% DMSO). 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.
Luciferase activity measurement: 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 was 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 luminometer to assess the quantity of luciferase (integration time one second).
Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (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 with the TECAN Infinite® M200 Pro Plate Reader.

Acceptability Criteria
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM (within two standard deviations of the historical mean of the testing facility). Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:

The results of the positive control are summarized in Table 2.
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.93 and the EC1.5 33.8 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.26 and the EC1.5 51.1 µM.

Run / experiment:

other: Exp 1

Parameter:

other: IC30

Remarks:

in µM

Value:

181

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Exp 1

Parameter:

other: IC50

Remarks:

in µM

Value:

249

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Exp 1

Parameter:

other: EC1.5

Remarks:

in µM

Remarks on result:

not determinable

Remarks:

No luminescence activity induction compared to the vehicle control was observed at non-toxic test concentrations

Run / experiment:

other: Exp 1

Parameter:

other: Imax

Value:

1.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

observed at toxic concentration with viability of 17.6%

Run / experiment:

other: Exp 2

Parameter:

other: IC30

Remarks:

in µM

Value:

306

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Exp 2

Parameter:

other: IC50

Remarks:

in µM

Value:

369

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Exp 2

Parameter:

other: EC1.5

Remarks:

in µM

Remarks on result:

not determinable

Remarks:

No luminescence activity induction compared to the vehicle control was observed at non-toxic test concentrations

Run / experiment:

other: Exp 2

Parameter:

other: Imax

Remarks:

in µM

Value:

0.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

• 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 in both experiments.
• The EC1.5 of the positive control was between 5 and 125 µM (33.8 µM and 51.1 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.93-fold and 2.26-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was with 8.0% below 20% in experiment 1 and 20.2% in experiment 2 (not deemed to affect study outcome because close to 20% and a clear negative effect of the test item was observed similar to the effect in experiment 1).

Historical control data: See Table 4

Table1: Overview luminescence induction and cell viability of CH02906 in Experiment 1
and 2

Concentration (µM)	0.977	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	0.92	1.12	1.19	1.34	1.24	1.31	1.38	1.29	0.94	0.50	0.90	1.54
Exp 1 viability (%)	103.7	97.4	100.2	93.6	87.2	92.2	91.7	86.2	49.8	19.0	36.2	17.6
Exp 2 luminescence	0.68	0.76	0.74	0.78	0.74	0.56	0.51	0.69	0.17	0.38	0.50	0.68
Exp 2 viability (%)	109.2	102.1	99.7	100.7	106.7	111.9	96.1	87.5	8.7	16.9	33.7	109.2

 

Table2: Overview luminescence induction and cell viability positive control Ethylene dimethacrylate glycol in Experiment 1 and 2

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	1.17	1.34	1.46	1.92^***	2.18^***	3.93^***
Exp 1 viability (%)	99.7	102.4	112.3	119.6	127.0	132.6
Exp 2 luminescence	0.80	1.03	1.52	1.63	2.26*	0.80***
Exp 2 viability (%)	100.7	110.6	113.6	119.0	113.1	100.7

^***p<0.001,^*p<0.05 Students t test

 

Table3: Overview EC_1.5, I_max, IC₃₀and IC₅₀values

	EC1.5 (µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.54	181	249
Test item Experiment 2	NA	0.78	306	369
Pos Control Experiment 1	33.8	3.93	NA	NA
Pos Control Experiment 2	51.1	2.26	NA	NA

N.A. = Not applicable

Table4: Historical Control Data for the KeratinoSens^TMStudies

	Positive control
	EC1.5	Imax
Range	5.7 – 109.0	1.79 – 8.20
Mean	42.6	3.34
SD	28.9	1.18
n	42	42

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2016 to March 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed toxicity (IC30 values of 181 µM and 306 µM and IC50 values of 249 µM and 369 µM in experiment 1 and 2, respectively). The maximum luciferase activity induction (Imax) was 1.54-fold (observed at 2000 µM with a viability of 17.6%) and 0.78-fold (observed at 7.81 µM with a viability of 100.7%) in experiment 1 and 2, respectively.
CH02906 is classified as negative in the KeratinoSensTM assay since only negative results (<1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.
Finally, it is concluded that the KeratinoSensTM assay is valid and that CH02906 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of CH02906to activate theantioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensÔassay.

The study procedures described in this report were based on OECD guideline 442D (February 2015).

Batch 151222 of CH02906 was a white to brownish powderwith a purity of 99.9%. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of0.977 – 2000 µM (2-fold dilution series). Two independent experiments were performed.

Acceptance criteria:

·       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 in both experiments. 

·       The EC_1.5of the positive control was within two standard deviations of the historical mean i.e. between 5 and 125 µM (33.8 µM and 51.1 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.93-fold and 2.26-fold in experiment 1 and 2, respectively).

·       Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was with 8.0% below 20% in experiment 1 and 20.2% in experiment 2).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test item showed toxicity (IC₃₀values of 181µMand 306µMand IC₅₀values of 249µMand 369µMin experiment 1 and 2, respectively). The maximum luciferase activity induction (I_max) was 1.54-fold (observed at 2000µMwith a viability of 17.6%) and 0.78-fold (observed at 7.81µMwith a viability of 100.7%) in experiment 1 and 2 respectively. 

CH02906is classified as negative in the KeratinoSens^TMassaysince only negative results (<1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

Finally, it is concluded that the KeratinoSens^TMassay is valid and that CH02906 is classified as negative(noactivation of theantioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Specific details on test material used for the study:

Smiles: C1(=C(C=CC(=C1)C#N)O)O

Run / experiment:

other:

Parameter:

other: EC3

Value:

0.06

Remarks on result:

other: QSAR Prediction

DEREK NEXUS version 5.0.2 yielded an alert for CH02906 for skin sensitization based on the presence of the 1,2-dihydroxybenzene derivative. CH02906 is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 0.060% (extreme sensitiser) based on nearest neighbours.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

QSAR prediction

Conclusions:

DEREK NEXUS version 5.0.2 yielded an alert for CH02906 for skin sensitization based on the presence of the 1,2-dihydroxybenzene derivative. CH02906 is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 0.060% (extreme sensitiser) based on nearest neighbours.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

9 October 2017

GLP compliance:

yes

Type of study:

activation of dendritic cells

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:

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

1. Test System:
Test System U937 human monocytes.
Justification Inducible CD86-expressing cells
Source ATCC (American Type Culture Collection, Virginia, USA).
ATCC no.: CRL-1593.2TM.
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. Once a year the cell line is checked for infection with a mycoplasma detection test.
Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

1.1. Vehicle
The vehicle of the test item, i.e. 0.4 % dimethyl sulfoxide (DMSO, Sigma, Zwijndrecht, The Netherlands) in complete medium.
1.2. Negative Control
Lactic Acid (LA, RS471) is used as negative control. On the treatment day, a solution at 10 mg/ml was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/ml stock solution (final dose level 200 µg/ml).
1.3. Positive Control
2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599) was provided as 1 M solution. On the treatment day a 10 mg/ml solution was prepared. This solution was diluted 1:100 in order to obtain a 0.1 mg/ml stock solution (final dose level 50 µg/ml).
1.4. Preparation of Test Item Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item formed a non-homogenous suspension in complete medium at 50 mg/mL. In DMSO the test item formed clear solution at 50 mg/ml.
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 in the 96-well plate (final concentration DMSO of 0.4%) in the first experiment and to a final test concentrations of 50, 20, 15, 10, 5 and 1 µg/mL in the second experiment.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test item concentrations were used within 1.5 hour after preparation.

2. Cell Culture
Cell culture medium:
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).

Environmental conditions:
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 80 –
99 %) containing 5.0 ± 0.5% CO2 (actual range 5.0 – 5.0 %) in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.4°C). Temperature and humidity were continuously monito red throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations of 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.

3. Experimental Design
3.1. Plating of Cells
Cultures were initiated in 96-well plates using 100 µl/well of a cell suspension adjusted at 5.0 x 10^5 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.
3.2. Number of experiments
At least two experiments were conducted per test substance to demonstrate reproducibility of the results and conclusion. If necessary, additional experiments were conducted in order to refine some equivocal/questionable findings. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 2 valid experiments were performed.
3.3. Treatment of Cells
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 six selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 1.0, 5.0, 10, 15, 20 and 50 µg/ml.
3.4. Precipitate evaluation
Before exposure and after 45 ± 3 hours of exposure, wells were checked for precipitate. If any, precipitate is documented in the raw data and reported in the table of the results.
3.5. Cell antibodies staining for IgG1 and CD86
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
- Human CD86 specific mouse IgG1
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.
3.6. Flow cytometry method
3.6.1. Acquisition
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 and the further analysis the BD FACSCanto™ flow cytometer was used.
3.6.2. Analysis
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.

3.6.3. Color Interferences
On IgG1 analysis
There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

On interferences of viability
There is colour interference in the viability when the X Median of the R2 window is 50% higher than the X Median of the vehicle control.

4. Acceptability Criteria
• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells is > 90%
• When DMSO is used as a solvent, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90%. The DMSO vehicle control is valid if the 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.
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
• No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
• The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
• Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150) and non-cytotoxic (cell viability ≥ 70%).

If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test was rejected and repeated.

Positive control results:

The positive control results with TNBS was valid

Run / experiment:

other: 1

Parameter:

other: EC150

Remarks:

unit: µg/mL

Value:

1.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

• A biologically relevant increase in the expression of CD86 was observed after treatment with CH02906, the EC150 is 1.8 µg/mL.

Run / experiment:

other: 2

Parameter:

other: EC150

Remarks:

unit: µg/mL

Value:

2.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• CH02906 showed toxicity, the calculated CV70 was 29 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with CH02906, the EC150 is 1.8 µg/mL.
Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• CH02906 showed toxicity, the calculated CV70 was 27 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with CH02906, the EC150 is 2.9 µg/mL.

Both tests passed the acceptance criteria:
• 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 99% 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% (4% in experiment 1 and 10% in experiment 2).
• 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 (first experiment, for second experiment see study plan deviation).

In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Discussion:
CH02906 showed toxicity to human histiocytic lymphoma cells (CV70 values of 29 µg/mL and 27 µg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 1.8 µg/mL and 2.9 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. CH02906 is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control for both experiments.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, CH02906 is classified as positive (an increase the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Executive summary:

The objective of this study is to evaluate the ability of CH02906 to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The study procedures described in this report were based on OECD guideline 442E (2017).

Batch151222of CH02906 was a white to brownish powder with a purity of 99.9%.  CH02906 was dissolved in dimethyl sulfoxide at 50 mg/mL. 

Two independent experiments were performed.In the first experiment the stock was diluted to six test concentration (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment, a more narrow dose-response analysis was performed to investigate the increase in expression of experiment 1 in more detail. In both experiments, no precipitate was observed at any dose level tested. 

Both experiments passed the acceptance criteria:

·     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 99% in experiment 2).

·     The DMSO vehicle control mean value of CD86 S.I. was smaller than 250% of the mean of CD86 S.I. of untreated U937 cells in both experiments.

·     The CD86 basal expression of untreated U937 cells was within the range of ≥ 2% and
≤ 25% (4% in experiment 1 and 10% in experiment 2).

·     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 (first experiment).

In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

CH02906 showed toxicity (CV70 values of 29 µg/mLand 27 µg/mLin experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 1.8 µg/mLand 2.9 µg/mLin experiment 1 and 2, respectively) was measured in both experiments. CH02906is classified as Positive in the U-Sens™ assaysince positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, CH02906 is classified as positive(anincrease the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

CH02906 gave a negative result in the DPRA assay and the KeratinoSens^TM assay. According to the DEREK NEXUS assessment, the test item should be considered to be a pre- or prohapten i.e. substances that require to be metabolized in order to act as skin sensitizers. CH02906 was predicted by DEREK to be oxidized into an electrophilic Michael acceptor in the form of a quinone. This explains why the DPRA and KeratinoSens^TMassay are negative as no metabolism system and minimal metabolism capability is present in these assays, respectively. The U-937 cell line in the U-Sens^TM assay has a different origin (lymphoma cells) with potentially more oxidizing capacity compared to the keratinocytes, as in this assay CH02906 is expected to have been metabolized to a quinone and consequently induced CD86 activity. Therefore, based on the positive DEREK and positive U-Sens^TM assay, CH02906 is considered to be a skin sensitizer.

In the DEREK NEXUS assessment a prediction for the EC3 value of 0.060% was given. This value was based on the 6 most closely related analogues, which were all 1,2-dihydroxybenzene derivatives, with EC3 values of 0.020 to 1.0%. Therefore, the prediction is considered sufficiently reliable to classify CH02906 in skin sensitization category 1A. No further in vivo testing is considered necessary.

In conclusion, CH02906 is considered to be a pre- or prohapten of a quinone by DEREK NEXUS, and the DPRA and KeratinoSens^TMassay are considered to be false negatives because they lack the potential for metabolism. The test item is considered a skin sensitizer based on the DEREK NEXUS assessment and the U-Sens^TMassay. The predicted value for EC3 of 0.060% classifies CH02906 in category 1A.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available