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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-24 - 2018-04-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

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

Version / remarks:

OECD Test Guideline 439 ,,OECD Guidelines for the testing of chemicals - In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method"(2015).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU-Method B.46 In vitro Skin Irritation: Human Epidermis Test Model, Annex to Commission Regulation (EU) No. 640/2012.

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human, not specified

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

Used in an in vitro testing strategy as first test in a bottom up approach.
Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in vivo. In contrast, organotypic skin explant systems are based on ex vivo skin removed from human or mouse and cultured in toto, afterwards. According to the literature all the models are useful in screening for topically applied irritant, corrosive or photocytotoxic substances

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.
The experiment was carried out on the reconstructed human epidermis model epiCS® (CellSystems, Troisdorf, Germany).
The tissue equivalents were shipped in 24 well cell culture plates on agarose supplemented with maintenance medium (Kit contents epiCS®, Cat.-No.CS-1001; epics® Frozen, Cat.No. CS-1001F; cellsystems). Inserts were of 0.6cm² size.
- Tissue batch number(s): Kit contents epiCS®, Cat.-No.CS-1001; epics® Frozen, Cat.No. CS-1001F; cellsystems

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Incubator temperature: 37 ± 2° C
The incubation of the treated epiCS inserts was carried out at RT for 20 min.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The inserts were washed carefully in PBS
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml in MTT-assay medium
- Incubation time: about 3 hours under cell culture conditions
- Spectrophotometer: automatic reader (EL808, Bio-Tek; 96 well format, 200 µl)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
each three inserts

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
• Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed control (inserts, which were killed by freezing) has to be used in the main assay
• Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
• Assessment of the color reaction with water
• Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control has to be used in the main assay.
The evaluation criteria for the pre-check are:
• For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
• For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
For interpretation of the cell viability results the cut-off percentage cell viability values distinguishing irritant from non-classified substances given in OECD TG 439 were used.
Table Evaluation criteria
In vitro result Evaluation
mean tissue viability ≤ 50 Irritant (Category 2 or Category 1)
mean tissue viability > 50 Non-irritant
Thus the test item is identified as irritant and requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤) to 50 %.
In case the test item was found to be non-corrosive (e.g. based on TG 431) and shows tissue viability after exposure and post-treatment incubation which is less than or equal (≤) to 50 %, the test item is considered to be irritant to skin in accordance with UN GHS Category 2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30µl
- Concentration (if solution): undiluted

VEHICLE : n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30µl
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30µl
- Concentration (if solution): 5 % SDS solution in NaCl 0.9%

Duration of treatment / exposure:

The incubation of the treated epiCS inserts was carried out at RT for 20 min.

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, mean value

Value:

96.68

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:

% tissue viability

Run / experiment:

positive control, mean value

Value:

1.62

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not irritating

Conclusions:

Testing was performed via a GLP OECD 439 guideline study on the registered substance itself. The present in vitro method is recommended in a tiered testing approach, it was used as the first tier in a bottom-up approach, which may serve as a standalone test in case no indication of irritation was observed.
The validity criteria are met, making the results sufficiently reliable to assess the irritating potential of the test item to the skin. The present in vitro method allows the identification of irritating chemical substances and mixtures. The test item Bis(2,4,6-triisopropylphenyl)carbodiimide did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item Bis(2,4,6‑triisopropylphenyl)carbodiimide with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

The study was conducted in accordance with OECD TG 439 and EU Test Method B.46 under GLP.

The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.

Cell viability was measured in a photometer by the amount 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 is set at 100%.

The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The following value of cell viability was recorded for the test item: 97 % (rounded).

In conclusion the results of the assay used show no skin irritant properties of the test item Bis(2,4,6-triisopropylphenyl)carbodiimide and thus, the test item requires no classification according to UN GHS .