reaction product of toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate) and aniline (molarratio 1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2017 - 09 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

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:

-

EC Number:

430-960-1

EC Name:

-

Molecular formula:

C21H20N4O2

IUPAC Name:

Reaction mass of aniline and m-tolylidene diisocyanate

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epithelial

Cell source:

other: human

Justification for test system used:

The EpiDerm™ Reconstructed Human Epidermis Model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 25855
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 07 November 2017
- Date of initiation of testing: 08 November 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item.
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: None reported
- Filter bandwidth: Not applicable
- Linear OD range of spectrophotometer: Not reported

NUMBER OF REPLICATE TISSUES: 2 per exposure period

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turns blue/purple relative to the control, the test item is presumed to have reduced the MTT. The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

An assessmemt of colour interference with the MTT endpoint was also made. 25 mg of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 or 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2 per exposure period

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean; 3 minutes exposure

Value:

93.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean; 60 minute exposure

Value:

86.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

Any other information on results incl. tables

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation	Relative Mean Variability
Negative Control	3 minutes	1.686	1.694	0.011	0.6	100*
		1.701
	60 minutes	1.803	1.831	0.040	2.2
		1.859
Positive Control	3 minutes	0.065	0.055	0.015	n.a.	3.2
		0.044
	60 minutes	0.038	0.038	0.001	n.a.	2.0
		0.037
Test Item	3 minutes	1.634	1.581	0.075	4.7	93.3
		1.528
	60 minutes	1.578	1.589	0.015	0.9	86.8
		1.599

n.a = not applicable

* the mean percentage viability of the negative control tissue is set at 100%

OD = Optical density

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of test item treated tissues after 3 and 60 minute exposure periods was 93.3 and 86.8 %, respectively. The test item was considered to be non-corrosive to skin.

Executive summary:

The skin corrosivity potential of the test item was assessed in an in vitro study Skin Corrosion: Reconstructed Human EpiDermis (RHE) test according to OECD 431 and EU B.40bis test guidelines alongside sterilised water and 8.0N potassium hydroxide as negative and positive controls, respectively. The mean viability of test item treated tissues after a 3 and 60 minute exposure period was 93.3 and 86.8 %, respectively. The test item was considered to be non-corrosive to skin.

 

The study is a GLP compliant guideline experimental study with no restrictions and therefore fully adequate for assessment for this endpoint.