Benzene-1,3-dicarbonitrile

Endpoint summary

Description of key information

Assessment of sensitisation via in-vitro methods.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 2019 - 21 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study, conducted to current gudeline.

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:

activation of keratinocytes

Justification for non-LLNA method:

According to Article 13 of the REACH Regulation and taking into consideration the testing strategies for the 2018 registration deadline (ECHA Endpoint Specific Guidance, Chapter R 7a, 2016), the information needed for the classification or risk assessment of a substance shall be generated whenever possible by means other than vertebrate animal tests, through the use of alternative methods.

An amendment to point 8.3 of Annex VII of the REACH regulation, (Commission Regulation (EU) 2016/1688 and Commission Regulation 2017/706 of 19 April 2017) requires that alternative methods are used for assessment of skin sensitisation potential where these will generate adequate information and where the available test methods are applicable to the substance to be tested.

Specific details on test material used for the study:

Name: Isophthalonitrile
Batch No.: JB1906150101
Chemical Name: 1,3-dicyanobenzene
CAS number: 626-17-5
EC number: 210-933-7
Molecular weight: 128.13 g/mol
Appearance: off white solid flakes
Expiry date: 1 5 June 2020
Purity: 99.8 % w/w
Storage: room temperature (15-25 ºC, below 70% relative humidity), protected from humidity (tight closed container)
Safety precautions: According to the SDS

Details on the study design:

KeratinoSens™ cell line
Name: KeratinoSens™ cell line
Description: immortalised adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids
Supplier: Givaudan Schweiz AG
Lot Number: 20160415
Date of production: April 15, 2016 [7]
Storage: Vapor phase of liquid nitrogen
The original cells (supplied by Givaudan upon establishing a license agreement) were propagated and subcultured into prepared cell line stocks (master cultures - MCs) in our laboratory. Cells from this original stock could be propagated up to maximum 25 passages and were employed for routine testing using the maintenance/growth medium.

Description of the Test

Procedure of the KeratinoSens™ method

0. Preincubation of cells
1. Seeding of cells for testing - 24 h incubation
2. Solubility assessment of the test item
Preparation of the stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement
6. Cytotoxicity assessment

Principle of the KeratinoSens™ method

The KeratinoSens™ method is an in vitro assay that quantifies the extent of luciferase gene induction following 48 hours incubation time of the KeratinoSens™ reporter cells with the test items. Luciferase gene induction is measured in the cell lysates by luminescence detection using a light producing luciferase substrate (Luciferase Reagent). Cytotoxicity and the relative luminescence intensity of luciferase substrate in the lysates are measured and luciferase induction compared to solvent/vehicle control is calculated.

KeratinoSens™ cells were derived from HaCaT human keratinocytes and transfected with selectable plasmids containing luciferase gene under the transcriptional control of the AKR1C2 ARE gene sequence, upstream of the SV40 promoter. AKR1C2 is known to be one of the genes up-regulated upon contacting skin sensitisers in dendritic cells. Therefore, this method is able to mimic the activation of the Keap1-Nrf2-ARE regulatory pathway, and is relevant for the assessment of the skin sensitisation potential of test items. A prediction model is used, to support the discrimination between sensitisers and non-sensitisers.

Preparation of the master plate

Test item Master Solutions

Based on the test item stock solutions made of DMSO, 2-fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test item creating a 100 × master plate. The 100 × master concentrations were further diluted 25-fold into exposure medium to obtain the 4 × master plate, by adding 10 µL of the 100 × master concentrations to 240 µL exposure medium.

Positive control

The positive control used was Trans-Cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.

Negative control

The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations in 6.3.1, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates.

This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.

Preparation of cells

Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.

Exposure

After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 µL of 4× master solution to 150 µL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.

Luciferase activity measurements

After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 µL), and 1× lysis buffer (20 µL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

Cytotoxicity

For the cell viability assay, medium was replaced after the 48-hour exposure time with MTT working solution (200 µL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 µL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 µM and 30 µM (11 µM and 7 µM in the first and second tests respectively)
The average inductions in the parallel plates for Trans Cinnamaldehyde at 64 μM were 6.86 fold and 19.12 fold in the first and second tests, respectively.
In the first test the luciferase activity induction was in the 2 – 8-fold induction range and there was a clear dose response relationship in the luciferase activity induction for the positive control.
Although the luciferase activity induction in the second test was outside of the 2 – 8-fold induction range, there was a clear dose response relationship in the luciferase activity induction for the positive control, therefore it was accepted as valid.

Key result

Run / experiment:

other: luciferase activity induction

Parameter:

other:

Value:

1.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: luciferase activity induction

Parameter:

other:

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

No precipitation was observed at any point during the first or second tests

Summary of the KeratinoSens™ results

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

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results and the KeratinoSens™ prediction model, the test item “Isophthalonitrile” was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In the course of this study the skin sensitization potential of the test item “Isophthalonitrile” was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. Since the results of the two runs were concordant, a third run was not needed to derive a conclusion.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests were the IC30 and IC50 values calculated.

Both tests were concluded negative, meaning that induction values for the test item did not exceed the 1.5-fold threshold, therefore EC1.5 values were not determined. Moreover, no dose response could be observed in any of the tests.

Based on these results and the KeratinoSens™ prediction model, the test item “Isophthalonitrile” was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 March 2020 to 13 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study, conducted to current gudeline.

Qualifier:

according to guideline

Guideline:

other: OECD 442E IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATION

Version / remarks:

OECD/OCDE 442E IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATION – Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) (25 June 2018)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Chemical induced expression of THP-1 cell surface markers CD86 and CD54 associated with the activation of dendritic cells (DC).

Specific details on test material used for the study:

No further details specified in the study report.

Details on the study design:

Test Concentrations of the Test Item
Test item concentrations for the main tests were determined in the dose finding (preliminary) tests.

As surrogate of human dendritic cells a human monocytic leukemia cell line, the THP-1 is used. Modulation of the expression of cell surface phenotypic biomarkers (CD86 and CD54) upon chemical exposure will be quantified by flow cytometric analysis of THP-1 cells. The h-CLAT method not only supports the discrimination between skin sensitisers from non-sensitisers, but it may also potentially contribute to the assessment of sensitising potency when used in integrated approaches such as the Integrated Approaches to Testing and Assessment (IATA).

Formulation of the Test Item
The solubility of the test item was tested in a non-GLP preliminary solubility test as follows:
First, the test item was tried to be formulated in sodium chloride solution (saline). At the concentration of 100 mg/mL the test item and the solvent separated into two phases even after 5 minutes of vortexing.
Then DMSO was tried at 500 mg/mL, but at this concentration only partial dissolution of the test item was observed after vortexing and undissolved particles were sank to the bottom of the test tube. With additional amount of DMSO, the test item was tried to be dissolved at 250 mg/mL, however still no total dissolution could be observed. By going down another 2 fold, at 125 mg/mL a total dissolution of the test item was observed with approximately 5 minutes of vortexing and a clear homogenous solution was formed.
Since the formulation with DMSO was suitable for the study, it was chosen as the appropriate solvent of the test item at the highest soluble concentration (HSC).

Positive control
Name: 1-CHLORO-2,4-DINITROBENZENE
Manufacturer/Supplier: SIGMA-ALDRICH
Batch: BCBZ3828
Expiry date: 22 May 2020
Storage: Room temperature
Purity: 99.8 %
CAS number: 97-00-7

Solvents/ Vehicles
Name: Dimethyl sulfoxide (DMSO)
Manufacturer: SIGMA-ALDRICH
Lot: RNBG7476
Expiry date: April 2020
Storage: Room temperature
CAS number: 67-68-5

Name: Sodium chloride solution (saline 0.9 %)
Manufacturer/Supplier: SIGMA-ALDRICH
Lot: RNBH2274
Expiry date: 18 April 2020
Storage: Room temperature
CAS number: 7647-14-5

Cell or Test System (THP-1 cell line)
The THP-1 cell line is an immortalized human monocytic leukemia cell line.

THP-1 stock
Name: THP-1
Description: Human Acute Monocytic Leukemia cell line
Supplier: ATCC - LGC Standards GmbH - Germany Office
ATCC Number: TIB-202
Lot Number: 63558119
Date of production: 09 MAY 2016
Storage: Vapor phase of liquid nitrogen
The original cells (TIB-202) were subcultured into prepared cell lines (master cultures-MCs) in our laboratory.

THP-1 Master culture
Subcultured MC2 master culture was used in the preliminary and main tests.
ID of the cell line: THP-1 (ATCC) MC2 20180205-20200131
Date of preparation: 05 February 2018
Date of thawing: 31 January 2020
Passage number at start: p8
Passage number at the end: p12
Reactivity check passed: 28 February 2020
Doubling time: ~ 41 hours

Maintenance (culture) medium for THP-1 cells
RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.05 mM L glutamine solution and 0.05 mM 2 mercaptoethanol.

Apparatus
Flow cytometer
Name: Apogee Flow Cytometer
Type: APOGEE A60 UNIVERSAL ANALIZER
Serial number: 0117
Laser (name, type): Laser Asbly A60 Univ 488nm, 50mW, Position C
Wavelength of the laser: 488 nm
Detectors / Filterblocks (488 nm): SALS, LALS
FL1:530/40
FL3:LP650

Evaluation softwares
Apogee Histogram Software for flow cytometry measurements and MS Excel for further calculations were used.

Preparation of Solutions
FACS (Fluorecence activated cell sorting) buffer: DPBS supplemented with 0.1 (w/v) % BSA.

100 × Blocking solution: 1 (w/v) % globulin Cohn fraction solution in DPBS.

1 × Blocking solution (0.01 (w/v) %): 100-fold dilution of 100 × Blocking solution with FACS buffer.

100 × PI solution: 1.25 mg/mL of propidium iodide solution in DPBS.

1 × PI solution (12.5 µg/mL): 100-fold dilution of the 100 × PI solution in DPBS.

FITC (Fluorescein isothiocyanate labelled) antibody solutions
(50 µL of pre-mixed solution per sample):
-FITC labelled CD86 antibody solution - 6 µL of antibody to 44 µL of FACS buffer
-FITC labelled CD54 antibody solution - 3 µL of antibody to 47 µL of FACS buffer
-FITC labelled-mouse IgG1 antibody solution - 3 µL of antibody to 47 µL of FACS buffer

Description of the Test
Principle of the h-CLAT Method
The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test item. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The activation process through which DCs change from antigen processing to antigen presenting cells addresses the third key event of the skin sensitization Adverse Outcome Pathway. The changes of surface phenotypic biomarker expression were measured and quantified by flow cytometry following cell staining with fluorochrome-tagged antibodies. The relative fluorescence intensity of surface markers compared to solvent/vehicle control were calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurred at sub-cytotoxic concentrations.

Demonstration of proficiency
Prior to routine use of the test method TOXI COOP ZRT. demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended in the OECD TG 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges. Moreover, a historical database of reactivity check results positive controls and solvent/vehicle controls was generated and has been maintained to confirm the reproducibility of the test method over time.

Procedure of the h-CLAT Method
0. Doubling time and reactivity check
Solubility assessment of the test item (formulation)
1. Preliminary tests (dose finding tests - CV75 determination)
2. Main Tests (CD86 and CD54 expression determination)

Doubling time and Reactivity check
Prior to using the master cell culture for testing, the cells were qualified by conducting a doubling time and reactivity check (approximately two weeks after thawing).
Doubling time was monitored following the instructions of the relevant SOP and the measured doubling time of approximately 41 hours fell into reference range of the EURL ECVAM DB-ALM protocol (30 h – 55 h). The generated data were introduced into a historical control database.
Reactivity check of the cells was performed using the positive controls: 1 chloro 2,4 dinitrobenzene (DNCB), nickel sulphate (NiSO4) and the negative control lactic acid (LA). The positive controls produced a positive response for both CD86 and CD54 marker expression, while the negative control produced a negative response for both CD86 and CD54 marker expression.
THP-1 (ATCC) MC2 20180205-20200131 master cell culture passed the reactivity check and concluded to be competent for the dose finding and main tests.

Preliminary tests
Preparation of the cells
For testing, THP-1 cells were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × 106 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 µL cell suspension / well (1 × 106 cells/well).

Preparation master and working solutions
Master solutions (MS) were prepared with DMSO as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution (highest soluble concentration - HSC), by two-fold serial dilutions using DMSO. These master solutions were then further diluted 250 fold into culture medium to obtain the working solutions (WS).
The working solutions were finally used for exposure by adding an equal volume of working solution (500 µL) to the volume of THP-1 cell suspension (500 µL) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item.

Solvent/vehicle control
The solvent/vehicle control used for the test item was 0.2 % DMSO and culture medium.

Test item exposure
The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated forn24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item.

Propium iodide (PI) Staining
After 24 ± 0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample.

Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.

Main Test (CD86 and CD54 expression)
Test item dilutions
The test item was first diluted to the concentration corresponding to the highest soluble concentration (HSC) determined in the solubility test and dose finding assay. DMSO was used to dissolve the test item for the stock solution (SS) in the main tests, as well. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using DMSO (eight concentrations). The master solutions were then further diluted 250-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.

Solvent/vehicle controls
Culture medium was used as negative control to assess the impact of DMSO. DMSO was tested as a solvent control for the test item and positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions.

Positive control
DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.

Application of test item and control substances
Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours.

Fluorescein Isothiocyanate (FITC) staining
After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at approximately 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes.
After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4° C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.

Positive control results:

The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each valid run.

Key result

Parameter:

other: h-CLAT result for CD86

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Parameter:

other: h-CLAT result for CD54

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Performance checks for cells (doubling time and reactivity check)
Before the preliminary tests, the above mentioned performance checks (doubling time and reativity check) were conducted for the cells.
THP-1 (ATCC) MC2 20180205-20200131 master cell culture passed the reactivity check and concluded to be competent for the dose finding and main tests.

Preliminary tests (Dose finding assays)
Two independent runs for the dose finding assay were performed to determine the test item concentration that results in 75 % cell viability (CV75) compared to the solvent/vehicle control chosen in the trial formulation.
In the first run the highest final test item concentration on the plate was 250 µg/mL and a 2 fold dilution was used when prepping the master solutions. Since the highest soluble concentration was used for the stock solution, no shift in the concentration range could be made even though no cytotoxicity was observed. In the second run, the same nominal final concentration on the plate and a 2-fold dilution was used.
Since no cytotoxicity was observed, no CV75 value was determined but the highest soluble concentration was used for setting the dose range for measuring CD86 and CD54 expression in the main test.

Main tests (CD86 and CD54 expression)
The CD86/54 expression was measured right after determining CV75, using the same batch of THP-1 cells.
For CD86/CD54 expression measurement, the test item was tested in four independent runs to derive a single prediction (positive/negative). Each independent run was performed on a different day or from a different batch/flask of cells. The last three out of the total four runs met the acceptance criteria therefore considered valid and only the valid runs were used in the prediction model.
The relative fluorescence intensity (RFI) was calculated for the test item, positive and negative controls for each concentration in each run for both surface markers, using the geometric mean fluorescence intensities.

Negative and positive control
The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each valid run.
The DMSO controls had negative outcomes compared to the medium control for both markers in all valid runs, meaning that the RFI values of CD86 and CD54 marker expression was never over the positive criteria. The cell viabilities of medium and DMSO controls were higher than 90 % in all valid runs (taken cell viabilities of the IgG1 isotypic control).
For medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control was over 105 % in all three valid runs.

Test item
The increase in CD86 marker expression (RFI) was greater than 150 % at at least one tested dose (with >50 % of cell viability) in each valid run, compared to the respective negative controls. However no clear dose response could be observed. Based on the three out of three positive results in the valid runs, CD86 marker expression was concluded to be concordantly positive.
The RFI values for CD54 expression were higher than 200 % at only one tested concentration in only one out of three independent valid runs, therefore only one run was concluded positive and two out of three runs were concluded negative.
Despite of the fact that CD54 marker expression was concluded to be negative, all three valid runs were positive for CD86 marker expressions, therefore the overall outcome of the study was positive.

Optional EC150 and EC200 values for sensitisers
Although concluded to be positive, no effective concentration (EC150) was determined for CD86 expression since no clear dose response could be observed in any of the valid runs.

Dose finding test results

Date	Test dose (μg/mL)	2.0	3.9	7.8	15.6	31.3	62.5	125.0	250.0
02-03 March, 2020	Viability (%)	96.3	96.0	96.6	95.9	96.5	96.5	95.4	96.3
Date	Test dose (μg/mL)	2.0	3.9	7.9	15.7	31.5	62.9	125.8	251.6
03-04 March, 2020	Viability (%)	95.9	96.3	96.5	96.1	95.9	96.4	96.3	96.0

 

Positive and negative control data

	Sample	Concentration	RFI		Viability (%) – IgG1
			CD86	CD54	IgG
Exposure date: 09 March, 2020	Medium	-	100	100	95.5
	DMSO	0.2%	101	82	95.3
	DNCB	4.0 μg/mL	548	846	73.7
Exposure date: 11 March, 2020	Medium	-	100	100	94.5
	DMSO	0.2%	132	73	94.7
	DNCB	4.4 μg/mL	543	854	77.7
Exposure date: 12 March, 2020	Medium	-	100	100	93.5
	DMSO	0.2%	122	109	94.1
	DNCB	4.2 μg/mL	606	604	76.4

 

Outcome of the individual valid runs of the main tests

Result of the individual runs for CD86 (positive/negative)				h-CLAT prediction for CD86 expression	Result of individual runs for CD54 (positive/negative)				h-CLAT prediction for CD54 expression
i	p	p	p	Positive	i	n	p	n	negative

p-positive outcome of a valid run

n-negative outcome of a valid run

i-invalid run

Interpretation of results:

study cannot be used for classification

Conclusions:

In the course of this study the skin sensitization potential of “Isophthalonitrile” was examined.

Based on the results and the h-CLAT prediction model, the test item “Isophthalonitrile” demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

In the course of this study the skin sensitization potential of “Isophthalonitrile” was examined.

 

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. Based on their result, eight doses between 250 µg/mL – 69.8 µg/mL were used for the main test in four independent runs out of which three runs were concluded valid. 

 

The increase in CD86 marker expression (RFI) was greater than 150 % at some tested doses (with >50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the three valid positive results out of three valid runs, CD86 marker expression was concluded to be positive. Effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed.

 

The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at only one concentration (with >50 % of cell viability) in the second valid run. Based on the two valid negative runs out of the three valid runs, CD54 marker expression was concluded to be negative.

 

Despite of the fact that CD54 marker expression was concluded to be negative, the overall h-CLAT prediction was concluded to be positive with three positive runs for CD86 marker expression.

 

Summary of the h-CLAT results for the test item

Obtained CV75 value (μg/mL)	h-CLAT result for CD86 (positive/negative)	h-CLAT result for CD54 (positive/negative)	h-CLAT result obtained (sensitizer/non-sensitizer)
Highest allowed concentration	Positive	Negative	Sensitizer

 

Based on the results and the h-CLAT prediction model, the test item “Isophthalonitrile” demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 December 2019 - 11 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

After the first run of analysis with cysteine and the evaluation of raw data, it could be concluded that the retention time of the test item was similar to the peptide and the area of the co-elution control was 1506 % of the area of the reference controls B and C. Thus Co-elution of the test item with the cysteine peptide was observed and the results could not be integrated into any of the prediction models (since there is no “only lysine model”). Knowing this information, the second run with lysine peptide had not been started. 
Based on the above mentioned result of the first run, the Direct Peptide Reactivity Assay for In Chemico determination of skin sensitisation potential with Isophthalonitrile was inconclusive and was subsequently terminated. The report is appended below.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Name: Isophthalonitrile
Batch No.: JB1906150101
Chemical Name: 1,3-dicyanobenzene
CAS number: 626-17-5
EC number: 210-933-7
Molecular weight: 128.13 g/mol
Appearance: off white solid flakes
Expiry date: 15 June 2020
Purity: 99.8 % w/w
Storage: room temperature (15-25 ºC, below 70% relative humidity), protected from humidity (tight closed container)

Details on the study design:

The solubility of the test item was tested in a non-GLP preliminary solubility test as follows: the solubility of the test item was evaluated at the concentration of 100 mM in several vehicles preferred by the guideline [3]. Several solvents from the guideline dissolved the test item completely (acetonitrile, acetone, 1:1 mixture acetone:acetonitrile, DMSO and acetonitrile in both given ratios).

However when the formulations were combined with the buffers of the test system (phosphate or acetate buffers and additional acetonitrile in case of phosphate buffer), in a ratio corresponding to the sample assembly description of the guideline, with acetate buffer we gained opalescent, precipitated solutions in all cases, but phosphate buffer worked fine with the formulations.

Therefore no solvent described in the guideline seemed to be suitable for the conduction of HPLC analysis under these conditions. However, the guideline suggests that if a precipitate or phase separation is observed, samples may be centrifuged at low speed (100-400xg) to force precipitate to the bottom of the vial as a precaution since large amounts of precipitate may clog the HPLC tubing or columns. Since a precipitate was observed immediately upon addition of the test chemical solution to the peptide solution, it cannot be sure how much test chemical remained in the solution to react with the peptide. Therefore, in such a case, a positive result could have been used, but a negative result not.

After centrifugation (400 g, 5 min) a clear supernatant could be easily removed, thus it could be used for HPLC analysis. Based on the preferred order of possible vehicles in the guideline, acetonitrile was chosen as the solvent for the test item, based on the observations above.

Key result

Remarks on result:

not determinable because of methodological limitations

Remarks:

See below

Interpretation of results:

study cannot be used for classification

Conclusions:

After the first run of analysis with cysteine and the evaluation of raw data, it could be concluded that the retention time of the test item was similar to the peptide and the area of the co-elution control was 1506 % of the area of the reference controls B and C. Thus Co-elution of the test item with the cysteine peptide was observed and the results could not be integrated into any of the prediction models (since there is no “only lysine model”). Knowing this information, the second run with lysine peptide had not been started.

Based on the above mentioned result of the first run, the Direct Peptide Reactivity Assay for In Chemico determination of skin sensitisation potential with Isophthalonitrile was inconclusive and was terminated.

Executive summary:

After the first run of analysis with cysteine and the evaluation of raw data, it could be concluded that the retention time of the test item was similar to the peptide and the area of the co-elution control was 1506 % of the area of the reference controls B and C. Thus Co-elution of the test item with the cysteine peptide was observed and the results could not be integrated into any of the prediction models (since there is no “only lysine model”). Knowing this information, the second run with lysine peptide had not been started. 

Based on the above mentioned result of the first run, the Direct Peptide Reactivity Assay for In Chemico determination of skin sensitisation potential with Isophthalonitrile was inconclusive and was subsequently terminated.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Three in vitro sensitisation assessments are available as follows:

h-CLAT

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. Based on their result, eight doses between 250 µg/mL – 69.8 µg/mL were used for the main test in four independent runs out of which three runs were concluded valid. 

The increase in CD86 marker expression (RFI) was greater than 150 % at some tested doses (with >50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the three valid positive results out of three valid runs, CD86 marker expression was concluded to be positive. Effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed.

The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at only one concentration (with >50 % of cell viability) in the second valid run. Based on the two valid negative runs out of the three valid runs, CD54 marker expression was concluded to be negative.

Despite of the fact that CD54 marker expression was concluded to be negative, the overall h-CLAT prediction was concluded to be positive with three positive runs for CD86 marker expression.

Based on the results and the h-CLAT prediction model, the test item demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

 

DPRA

After the first run of analysis with cysteine and the evaluation of raw data, it could be concluded that the retention time of the test item was similar to the peptide and the area of the co-elution control was 1506 % of the area of the reference controls B and C. Thus Co-elution of the test item with the cysteine peptide was observed and the results could not be integrated into any of the prediction models (since there is no “only lysine model”). Knowing this information, the second run with lysine peptide had not been started. 

Based on the above mentioned result of the first run, the Direct Peptide Reactivity Assay for In Chemico determination of skin sensitisation potential with Isophthalonitrile was inconclusive and was subsequently terminated.

 

ARE-Nrf2 Luciferase Test Method

For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. Since the results of the two runs were concordant, a third run was not needed to derive a conclusion.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests were the IC30 and IC50 values calculated.

Both tests were concluded negative, meaning that induction values for the test item did not exceed the 1.5-fold threshold, therefore EC1.5 values were not determined. Moreover, no dose response could be observed in any of the tests.

Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Conclusion

Based on the results observed (one negative, one positive and one disregarded) within the in-vitro tests, the test item is classified as a skin sensitiser, category 1 as a worst case assessment.