Benzene-1,3-dicarbonitrile

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2019 - 05 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study to current guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: 15 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

Test system:

human skin model

Source species:

other: In-Vitro EpiSkin Model

Cell type:

other: Adult human-derived epidermal keratinocytes

Cell source:

other: Manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model.

Source strain:

other: Manufactured by EPISKIN SNC Lyon, France

Details on animal used as source of test system:

No animals used as this is an In-Vitro test test.

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Pre-incubation
The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere.

Application
Before application the skin units were placed into pre-warmed “assay medium” (37°C; 2 mL per well).
Two replicates were used for the test item and control(s) respectively.

Test Item
An amount of 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Positive and negative control
A volume of 50 µL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly the complete epidermal surface if necessary.

Exposure
The plates with the treated epidermis units were incubated for the exposure time of 4 hours (±10 min) at room temperature (23.2-24.4 °C).

Rinsing
After the incubation time the EpiSkinTMSM units were rinsed thoroughly with approximately 25 mL 1x PBS solution to remove the test item from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT test

To terminate the exposure with the test item, the EpiSkinTMSM units were rinsed with PBS. Thereafter, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±1 °C in an incubator with 5±1 % CO2 protected from light, ≥95 % humidified atmosphere.

Formazan extraction

At the end of incubation with MTT a formazan extraction was undertaken:

A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

Cell viability measurements

Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2908 – 2.6589) using acidified isopropanol solution as the blank (6×200 µL.

Calculations of viability percentages

The calculations were performed using Microsoft Excel software.
Blank:
The mean of the 6 blank OD values was calculated.

Negative control:
Individual negative control OD values were corrected with the mean blank OD:
OD Negative Control (ODNC1) = ODNCraw1 – ODblank mean
OD Negative Control (ODNC2) = ODNCraw2 – ODblank mean
Mean OD Negative Control (mean ODNC) = [(ODNC1) + (ODNC2)] / 2
The corrected mean OD of the 2 negative control values was calculated: this corresponds to 100 % viability.

Positive control:
Individual positive control OD values were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
The corrected mean OD of the 2 positive control values was calculated
The % viability for each positive control replicate was calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100

The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (% PC1 + % PC2) / 2
Test item:
– Individual test item OD values were corrected with the mean blank OD:

OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
The corrected mean OD of the 2 test item values was calculated
The % viability for each test item replicate was calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
The mean value of the 2 individual viability % for test item was calculated
Mean TT % = (% TT1 + % TT2) / 2

Assay Acceptance Criteria

The mean OD value of the two negative control tissues should be between 0.6 and 1.5.
The acceptable percentage viability for positive control (each of two tissues) is
0 – 20 % (as per the manufactures specification, validated by ECVAM (Fentem et al, 1998)).
In the range 20-100 % viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30 %.

Interpretation of test results
The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” updated December 2011 / February 2012.
The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem et al, 1998).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

skin units/exposure times respectively. Subsequently, 100 µL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

4 hours (±10 min)

Number of replicates:

2 replicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other:

Remarks:

Optical Density

Run / experiment:

1

Value:

0.865

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other:

Remarks:

Optical Density

Run / experiment:

2

Value:

0.956

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item:
The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is off white and therefore considered not to be able to significantly stain the tissues and lead false estimate of viability, Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Any other information on results incl. tables

Cell viability

 

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

 

Table 3:OD values and cell viability percentages of the positive and negative control:

Controls	Optical Density (OD)		Viability (%)	Δ%
Negative Control: NaCl (9 g/L saline)	1	1.037	105	10.1
	2	0.937	95
	Mean	0.987	100
Positive Control: Glacial acetic acid	1	0.078	8	5.1
	2	0.028	3
	Mean	0.053	5

 

Table 4: OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)	Δ%
Isophthalonitrile	1	0.865	88	9.2
	2	0.956	97
	mean	0.910	92

 

Remark:     

1.    Mean blank value was 0.0402

2.    Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3.    Δ%: The difference of viability between the two relating tissues

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours (±10 min) at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.
NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively.
For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.
The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 92 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure.
Positive and negative controls showed the expected cell viability values within acceptable limits.
All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model (OECD 431), indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Isophthalonitrile can be classified as Non-corrosive to skin.

Executive summary:

Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours (±10 min) at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO₂in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 92 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits.

All assay acceptance criteria were met, the experiment was considered to be valid.

 

The results obtained from this in vitro skin corrosion test, using the EPISKIN model (OECD 431), indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Isophthalonitrile can be classified as non-corrosive to skin.