Benzene-1,3-dicarbonitrile

Endpoint summary

Description of key information

Skin and eye irritation in vitro are assessed.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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

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

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

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.

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

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2019 - 06 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study to current guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

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 for this In-Vitro test used.

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System

Human Skin

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.

Supplier: EPISKIN Laboratories
4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 19-EKIN-049
Expiry date: 09 December 2019

Justification for selection of the test system

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Quality Control

EpiSkinTMSM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).
 
EpiSkinTMSM KIT Contents

Units: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations.
(Batch No.: 19-MAIN3-057; Exp. Date: 11 December 2019)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 19-ESSC-051; Exp. Date: 11 December 2019)

EpiSkinTMSM KIT Reception Procedure

The colour of the agar medium used for transport was checked for its pH:
- orange colour = good
- yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C:
- the indicator changes from white to grey at 40 °C

The kit was found to be in good order at reception.

EpiSkinTMSM KIT Storage Procedure

The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8 °C.

Indicator for potential false viability

Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.
 
Check-method for possible direct MTT reduction with test item

Approximately 10 mg test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C protected from light and then any colour change observed:
- Test items which do not interact with MTT: yellow
- Test items interacting with MTT: blue or purple

If the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).

Check-method to detect the colouring potential of test item

Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
Approximately 10 mg test item was added to 90 μL of water (prepared in Toxi-Coop ZRT. by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C) and mixed. The mixture was shaken for 15 minutes at room temperature and then colour checked (unaided eye assessment).

Test Procedure

Pre-incubation (day [-1]-0)

The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of an 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, ≥95 % humidified atmosphere.

Application (day 0)

Three replicates were used for the test item and positive and negative controls, respectively.

Test Item
The epidermal surface was first moistened with 5 µL deionised water* in order to improve further contact between powder and epidermis. Subsequently, approximately 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
* prepared by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C in Toxi-Coop ZRT.

Positive and negative control
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Exposure (day 0)

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (24.0 – 24.4°C).

Rinsing (day 0)

After the incubation time, the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material 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).

Post-incubation (day 0-2)

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1 h) at 37±1 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere.

MTT test after 42 hours incubation (day 2)

After the 42 hours (± 1h) incubation, 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 (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95 % humidified atmosphere.

Formazan extraction (day 2)

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 for approximately four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

Cell viability measurements (day 2)

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).
 
Evaluation of Experimental Data

Calculations of viability percentages

Data calculation for normal test items

Blank:
– The mean of the 6 blank OD values is calculated
Negative control:
– Individual negative control OD values are corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 3 negative control values is calculated: this corresponds to 100% viability
Positive control:
– Individual positive control OD values are corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values is calculated
– The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item:
– Individual test item OD values are corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test item values is calculated
– The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3
 
Assay Acceptance Criteria

The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

Interpretation of test results

According to the United Nations Globally Harmonized System (UN GHS) of Classification and Labelling of Chemicals (8th revised edition; 2019) and as implemented in the European Commission Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (EU CLP), the irritancy potential of test substances is predicted for distinguishing between irritant or corrosive (Category 2 or Category 1) and non-irritant (No Category) substances.
In the present study, the irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion (OECD 431) will be required to decide on its final classification. In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 mg of the test item was applied evenly to the epidermal surface

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 h) at 37±1 °C

Number of replicates:

3 replicates

Irritation / corrosion parameter:

other:

Remarks:

Optical Density

Run / experiment:

1

Value:

0.839

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other:

Remarks:

Optical Density

Run / experiment:

2

Value:

0.945

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: Optical Density

Run / experiment:

3

Value:

1.096

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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:

 

OD values and viability percentages of the controls and test item:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.887	98
	2	0.796	88
	3	1.022	113
	mean	0.902	100
	standard deviation (SD)		12.58
Positive Control: SDS (5 % aq.)	1	0.037	4
	2	0.103	11
	3	0.060	7
	mean	0.067	7
	standard deviation (SD)		3.68
Test Item: Isophthalonitrile	1	0.839	93
	2	0.945	105
	3	1.096	122
	mean	0.960	106
	standard deviation (SD)		14.34

 

Remark:     

Mean blank value was 0.040

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

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Isophthalonitrile is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

EpiSkin^TMSM test of Isophthalonitrile has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,18 June 2019.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes
(± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO₂,≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO₂protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

In thisin vitroskin irritation test using the EPISKIN model, the test item Isophthalonitrile did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Isophthalonitrile is considered to be non-irritant to skin and is therefore not classified(UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2019 - 20 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study to current guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

chicken

Strain:

other:

Remarks:

COBB 500 (1) and ROSS 308 (2)

Details on test animals or tissues and environmental conditions:

Strain of chicken: COBB 500 (1) and ROSS 308 (2)
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary

Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3ºC to 20.4ºC in the first and 19.2ºC to 20.4ºC additional experiment) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

Identification
The eyes were identified by chamber number, marked on the door of the chamber.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: Fluorescein sodium salt

Amount / concentration applied:

The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control.

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Not applicable for In-Vitro study

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control treated eye which was treated with 30 µL saline solution were used in the first and additional experiment.

Details on study design:

Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes during the first and additional experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet, if applicable. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

Test procedure

Treatment
After the zero reference measurements in each experiment, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.
The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

Test item removal
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

The Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and additional experiment.

Observation and assessment of corneal effects
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.

The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

Retention of corneas
At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

Histopathology
A histopathology of the corneas was not performed. Corneas are discarded 2 months after the final report.

Demonstration of Proficiency
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity

Value:

> 0.5 - < 1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 75 min

Value:

> 3 - < 5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 240 min

Value:

> 4 - < 5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean fluorescein retention

Value:

> 0.2 - < 0.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.
Positive and negative control values were within the corresponding historical control data ranges

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below.

 

Test Item: Isophthalonitrile

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3%	I
Mean maximum corneal swelling at up to 240 min	4%	I
Mean maximum corneal opacity	0.8	II
Mean fluorescein retention	0.2	I
Other Observations	None
Overall ICE Class	2xI, 1xII

 

Positive Control: Imidazole

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	31%	IV
Mean maximum corneal swelling at up to 240 min	36%	IV
Mean maximum corneal opacity	3.8	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in three eyes eye at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

 

The positive control Acetic acid 10% (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

 

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.

 

Additional experiment:

 

Test Item: Isophthalonitrile

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	5%	I
Mean maximum corneal swelling at up to 240 min	5%	I
Mean maximum corneal opacity	1.0	II
Mean fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class	2xI, 1xII

  

Positive Control: Imidazole

 

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	30%	IV
Mean maximum corneal swelling at up to 240 min	36%	IV
Mean maximum corneal opacity	3.7	IV
Mean fluorescein retention	2.7	IV
Other Observations	Corneal opacity score 4 was observed in two eyes and score 3 was seen in one eye at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

The positive control Acetic acid 10% (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Interpretation of results:

GHS criteria not met

Conclusions:

In this ICET, Isophthalonitrile did not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either cases. The overall ICE class was 2xI, 1xII in the first and the additional experiment.
The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative control were in the historical control range. So the positive and negative controls showed the expected results. The experiment was considered to be valid in both occasions.

In this in vitro eye irritation study, using the Isolated Chicken Eye model with Isophthalonitrile, no ocular corrosion or severe irritation potential was observed. The overall ICE class was 2xI, 1xII in the first and the additional experiment.
According to the guideline OECD 438, Isophthalonitrile is categorized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Isophthalonitrile by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

 

According to the first experiment results the test item showed negative outcome (GHS NC). As it was a solid test item, according to the OECD 438 additional experiment was necessary to confirm or discard the negative outcome.

 

The Imidazole (positive control) and test item Isophthalonitrile was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes, three positive control treated eyes and one negative control treated eye which was treated with 30 µL saline solution were used in the first and additional experiment.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

 

The Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and additional experiment. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and additional experiment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion:

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.

 

 

Skin irritation:

EpiSkinTMSM test of Isophthalonitrile has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,18 June 2019.

 

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2,≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

 

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

 

In thisin vitroskin irritation test using the EPISKIN model, the test item Isophthalonitrile did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

 

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Isophthalonitrile is considered to be non-irritant to skin and is therefore not classified(UN GHS No Category).

Eye Irritation:

The Isolated Chicken Eye Test (ICET) was used to evaluate the potential ocular corrosivity and irritancy of the test item Isophthalonitrile by its ability to induce toxicity in enucleated chicken eyes.

According to the first experiment results the test item showed negative outcome (GHS NC). As it was a solid test item, according to the OECD 438 additional experiment was necessary to confirm or discard the negative outcome.

The Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinsein the first and additional experiment. The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinsein the first and additional experiment.
In this ICET, Isophthalonitrile did not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either cases. The overall ICE class was 2xI, 1xII in the first and the additional experiment.

In this in vitro eye irritation study, using the Isolated Chicken Eye model with Isophthalonitrile, no ocular corrosion or severe irritation potential was observed. The overall ICE class was 2xI, 1xII in the first and the additional experiment.

According to the guideline OECD 438, Isophthalonitrile is categorized as “No Category”.