N,N'-bis(2,4,6-triisopropylphenyl)methanediimine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-02-14 - 2018-02-15 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

OECD 492

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-08 - 2018-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Testing Guideline 492 (Adopted 09 Oct 2017) for the testing of chemicals: Reconstructed human cornea-like epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.

Deviations:

no

GLP compliance:

yes

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Test System/ Study Design
The Epiocular™ RhCE tissue construct consists of 3 viable layers of cells and a nonkeratinized
surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in Epiücular™ EIT is measured by enzymatic conversion ofthe vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution): undiluted

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

12 min + 120 min

Number of animals or in vitro replicates:

duplicates

Details on study design:

- Details of the test procedure used
This study was performed in compliance withOECD Testing Guideline 492 (Adopted 09 Oct 2017) for the testing of chemicals: Reconstructed human cornea-like epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.
- RhCE tissue construct used, including batch number
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium (Part#: OCL-200; Lot No.: 27027; Testing date: 14 Mar 2018).
- Doses of test chemical and control substances used
50µl
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Upon receipt the tissue inserts were allowed to equilibrate to room temperature for 15 minutes. Subsequently each tissue insert was transferred from the packaging plate into the well of a 6 well culture plate containing 1ml of fresh prewarmed (approx. 37°C) maintenance medium. The EpiOcular™ inserts were incubated for 1 hour under SCC. Afterwards a medium change was performed and the tissue inserts were allowed to further adapt overnight to the recommended SCC.
The culture conditions in the incubator were standardized as follows:
Incubator temperature 37 ± 2°C
CO2 gas concentration 5 %
Humidity 95 %
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door. However, these deviations had no effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode-Germany).
Treatment: 50 µl of the neat test item, negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate. The tissues were treated for about 30 minutes under SCC. After the end of treatment time the test item is removed by rinsing with PBS followed by a post-soak immersion period of about 12 min. in fresh medium.
Post-treatment incubation: the tissues were incubated for 120 ± 5 min. in fresh medium under SCC.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
Killed control and color control not required.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
duplicate
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) / Description of the method used to quantify MTT formazan
The MTT assay is a standardized quantitative method that is used to measure tissue viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3 [4,5 Dimethythiazol 2 yl] 2,5 diphenyl tetrazolium bromide (MTT) (Sigma, Deisenhofen-Germany) to blue formazan crystals.
Immediately after the end of post-treatment incubation the MTT reduction assay was performed. For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C, or Assay medium for the color control inserts). The tissues were incubated for 180 ± 10 min. under SCC. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µl).
Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).
The OD values obtained with the replicate tissue extracts for the test item were used to calculate the mean percent tissue viability normalized to negative control, which was set at 100 %.
The final viability value for the test item was then calculated by subtracting the appropriate controls from the test item treated values (color control (CC) / killed control (KC)) and added the Non-specific killed control NS KCas follows:
Final viability test item = % viability test item – (%viability test item KC - % viability NC KC) - % viability test item CC + viability NS KC.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Assay Acceptance Criteria
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.
Interpretation of Results and Evaluation Criteria
For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.
- Acceptable variability between tissue replicates for positive and negative controls: met
- Acceptable variability between tissue replicates for the test chemical: met

Irritation parameter:

other: % cell viability

Run / experiment:

mean

Value:

90.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

cell viability 7.4%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Interpretation of results:

GHS criteria not met

Remarks:

No GHS category

Conclusions:

The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro. As the final test item-treated tissue viability was > 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS No Category).
The present study was conducted as a follow-up study to the preliminarily conducted BCOP assay, in which no prediction could be made. So taking into account the present result, the substance does not need to be classified as irritating to the eye.

Executive summary:

An in vitro study for assessing ocular irritation properties of the test item Bis(2,4,6-triisopropylphenyl)carbodiimide was performed under GLP using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM. The model used is standardized and commercially available.

The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492.

The undiluted test item was applied topically to the RhCE tissue surface in duplicate for 30 minutes, followed by a 120 minutes post-treatment incubation period.

Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model.

The final mean percent tissue viability recorded for the test item is 90 % (rounded).

According to the results of this study the test item Bis(2,4,6-triisopropylphenyl)carbodiimide was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Test Guideline 437 - “OECD Guideline for the Testing of Chemicals - Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”, 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

EU method B. 47. Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Annex to Commission Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[2,4,6-tris(propan-2-yl)phenyl]methanediimine

EC Number:

815-171-4

Cas Number:

300382-79-0

Molecular formula:

C31H46N2

IUPAC Name:

N,N'-bis[2,4,6-tris(propan-2-yl)phenyl]methanediimine

Test material form:

liquid

Remarks:

clear yellow

Details on test material:

Storage: Room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3 corneas

Details on study design:

SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE ISOLATED CORNEAS
Extraction: Staff of the slaughterhouse
Transport: 1 L containers with 500 mL HSS and 1 % penicillin / streptomycin solution, transport of the containers in coolers on ice.
The test system matches with the guideline
Preparation of Corneas:
Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter. Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight, which is not compromising the assay results, at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
On the next day the containers with the eyes were transferred in an incubator at 32 °C (± 1°C) for about 2 hours.
For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour in the incubator at 32 °C (± 1 °C).
Selection of corneas for application
After approximately 1 hour, the MEM medium was aspirated and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± 1 standard deviation were selected for the actual test and assigned to the test groups.
The numbers of the selected corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.

NUMBER OF REPLICATES
Triplicates

NEGATIVE CONTROL USED
750 µl isotonic saline solution

POSITIVE CONTROL USED
750 µl 1 % NaOH in isotonic saline solution (w/v), clear solution

APPLICATION DOSE AND EXPOSURE TIME
750 µl, 10 min

TREATMENT METHOD:
Immediately before application, the medium was aspirated from the anterior chamber. 750 μL of negative and positive control and of the test item were applied to the corneas through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the liquids covered the cornea sufficiently. The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 10 minutes.

POST-INCUBATION PERIOD: yes, 2h

REMOVAL OF TEST SUBSTANCE / POST-EXPOSURE INCUBATION:
After the exposure, the negative and positive control and the test item were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea.
Before further incubation, the medium was aspirated from both chambers, fresh MEM medium was filled into the chambers. The holders were incubated for additional approx. 2 hours at 32 °C (± 1 °C).
Before measuring opacity, fresh MEM medium was filled in the chambers.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.
The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort V3.4 SP6 software from Testo AG, Lenzkirch). The validation of the opacitometer was carried out under the study number T8082017.
Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1 mL of fluorescein sodium solution (concentration 4 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes.
After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 4 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader (Bio-Tek EL 808, Software Gen5).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Calculation and Evaluation of In Vitro Irritancy Score (IVIS)
All parameters and the IVIS values were calculated by using Microsoft Excel. In the tables of the Annex individual values have been rounded. In the calculation of means and variances etc., original non-rounded values were taken as the basis in some cases.
The validation of the Excel file was carried out under the study number T0082019.
The opacity values were calculated by applying the following formulae:
1) Opacity = (I0/I-0.9894)/0.0251
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity change = opacity change - mean opacity change NC
4) Mean opacity = mean of all corrected opacity changes per group
The permeability values were calculated by applying the following formulae:
1) OD490 change = OD490 value - mean blank value OD490
2) Corrected OD490 change = OD490 change - mean OD490 change NC
3) Mean OD490 = mean of all corrected OD490 changes per group
Calculation of In Vitro Irritancy Score (IVIS):
1) IVIS per cornea = corrected opacity change + (15 x corrected OD490 change)
2) IVIS per group = mean of IVIS values per cornea in a group
I0 = single value of the measurement of empty holder with medium but without cornea, measured 1-2 days before the experiment.
I = individual value of each opacity measurement before and after application
0.9894 / 0.0251 is a constant, which is required for calculation.

DECISION CRITERIA: The decision criteria as indicated in the TG was used.
The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

Table Decision criteria
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made *
> 55 Category 1
*: In case “no prediction can be made” classification as Category 1 is not warranted. This would be indicated as “No Category 1”.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS = 2.0

Positive controls validity:

valid

Remarks:

IVIS = 157.7

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Follow-up OECD 492 study is conducted

Conclusions:

The study was conducted under GLP according to OECD guideline 437 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.
Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category 1). However, a differentiation between Cat. 2 or no classification cannot be made. So a follow-up study acc. OECD TG 492 is performed.

Executive summary:

This GLP study was performed to assess the corneal damage potential of the liquid test item Bis(2,4,6-triisopropylphenyl)carbodiimide with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.

The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

The undiluted test item and the positive control 1 % sodium hydroxide in isotonic saline solution were tested on 3 bovine corneas each in comparison to the negative control, isotonic saline solution. For determination of corneal damage opacity as well as tissue permeability was measured after a 10 minute treatment time.

Test items were applied to the epithelial surface of the cornea in a special corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. The In Vitro Irritancy Score (IVIS) value was calculated based on these data.

In accordance with OECD TG 437 and the study results Bis(2,4,6-triisopropylphenyl)carbodiimide was characterized by having no potential to seriously damage the eye.

 

Table Classification of test item
Test item	In vitro irritancy score (IVIS)	Classification
Bis(2,4,6-triisopropylphenyl)carbodiimide	4.0	No Category 1