Benzene-1,3-dicarbonitrile

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

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.

Test material

Test material information

Constituent 1

Reference substance name:

Benzene-1,3-dicarbonitrile

EC Number:

210-933-7

EC Name:

Benzene-1,3-dicarbonitrile

Cas Number:

626-17-5

Molecular formula:

C8H4N2

IUPAC Name:

benzene-1,3-dicarbonitrile

Test material form:

solid: flakes

Details on test material:

Test substance name: Isophthalonitrile
Chemical name: 1,3-dicyanobenzene
CAS number: 626-17-5
EC number: 210-933-7
LOT number: JB1906150101
Purity: 99.8 % (w/w %)
Appearance: Off white solid flakes
Manufacture date: 15 June 2019
Expiry date:15 June 2020
Storage condition: Room temperature (15-25oC. below 70 RH%), protected from humidity

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

In vitro test system

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.

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).