2,4,6-trimethylbenzene-1,3-diamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-02 to 2016-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

adopted July 28, 2015;

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 06, 2012.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trimethylbenzene-1,3-diamine

EC Number:

221-456-9

EC Name:

2,4,6-trimethylbenzene-1,3-diamine

Cas Number:

3102-70-3

Molecular formula:

C9H14N2

IUPAC Name:

2,4,6-trimethylbenzene-1,3-diamine

Test material form:

other: crystalline powder

Details on test material:

2,4,6-trimethylbenzene-1,3-diamine supplied by Evonik Fibres GmbH (Lenzing, Austria), batch 20150703 (Dragon), purity 99.82 % w/w

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be
soluble or insoluble in water.

Vehicle:

other: Dulbecco’s phosphate buffered saline.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:The following Reconstructed Human Epidermis Model was used: EpiDermTM (EPI-200, Lot no. 23338) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- The test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 6.4
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination.
- MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot.
- The ET50 must fall within a range established based on a historical database of results.

NUMBER OF REPLICATE TISSUES: Three replicate tissues were employed

TEST FOR INTERFERENCE OF TEST ITEM WITH MTT REDUCTION ASSAY
- Prior to the testing, the test item was evaluated of colour changes under aqueous conditions. Therefore, 25 mg test item was diluted in
300 µL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolorations were noted.
- Furthermore the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 25 mg test item was
dissolved in 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as
control. No change of colour was noted.
Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

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



ADMINISTRATION
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
- The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.
- Three replicate tissues were employed
- At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- The whole exposure period for the used EpiDermTM skin model was 60 minutes
- The incubation conditions were 37°C, 5% CO2 and 95% humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue
sensitivity (PC) of the tissues are within a defined historical acceptance range
- Positive control item was 5% aqueous sodium dodecyl sulphate (SDS)
- Negative control was D-PBS5
- 30 μL of negative and positive controls were used

- Viability measurements were not performed immediately after the exposure to the test item, but after a post-treatment incubation period of the
rinsed tissues in fresh medium of 42 hours.
- This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects
- Each skin sample was placed in an MTT solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours
- The precipitated blue formazan product was extracted using the extraction solution (isopropanol)
- Concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer
(Tecan Sunrise Magellan Version 6.4)
- Measurements were made for each of the three tissues in 2 replicates

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
- The test item is a fine powder.
- For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.
- Three replicate tissues were employed.
- At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

VEHICLE
- Dulbecco's phosphate buffered saline (D-PBS)

NEGATIVE CONTROL
- 30 µL Dulbecco's phosphate buffered saline (D-PBS)

POSITIVE CONTROL
- 30 µL 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

An exposure time of 60 minutes was employed.

Duration of post-treatment incubation (if applicable):

Post-treatment incubation period of the rinsed tissues in fresh assay medium of 42 hours.

Number of replicates:

Three replicate tissues were employed.

Test animals

Species:

other: in vitro

Test system

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

60 minutes

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: cut-off percentage cell viability

Run / experiment:

The test method is based on reconstructed human epidermis models

Value:

39.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cytotoxic and irritant to skin

Other effects / acceptance of results:

Test for interference of chemicals with MTT
Optical properties of the test item or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability, as the test item may prevent or reverse the colour generation as well as cause it. This may occur when a specific test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.

Prior to the testing, the test item was evaluated of colour changes under aqueous conditions. Therefore, 25 mg test item was mixed with 300 µL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolorations were noted.

Furthermore, the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 25 mg test item were mixed with 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No change of colour was noted.

Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.


Assay acceptability criteria

Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.

Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.

Assay acceptance criterion 3: Standard deviation
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18%

Any other information on results incl. tables

Interpretation of results

The OD values obtained for each test sample were used to calculate mean percentage viability relative to the negative control, which is set at 100%. The cut-off mean percentage cell viability value that distinguishes irritant from non-classified test substances is given below:

According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.

mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS category 1 or 2)

mean tissue viability > 50% non-irritant (NI).

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties of the test item to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensionalmodel of human skin. TheEpiDerm^TMmodel according to OECD 439 was employed.

 

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Test item was applied topically as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

 

The mean viability of cells exposed to test item was 39.2% of the negative controls and, hence, was below the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of ≤ 50%. Test item was considered to be cytotoxic and predicted to be irritant to skin in accordance with UN GHS category 2. As test item was found to be non-corrosive in an in vitro skin corrosion test (LPT Report No. 33340) according to OECD guideline 431 distinguishing between Category 1 or Category 2 is admissible.

 

The mean optical density (OD) of the negative control of 3 tissues each was 1.417 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 9.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.

The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria required (see section 6.6) were fulfilled.

 

Conclusion

Under the present test conditions, test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.