2,4,6-trimethylpyridine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-12-19 to 2020-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trimethylpyridine

EC Number:

203-613-3

EC Name:

2,4,6-trimethylpyridine

Cas Number:

108-75-8

Molecular formula:

C8H11N

IUPAC Name:

2,4,6-trimethylpyridine

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™, Epi-200-SIT kit and MTT-100 assay, purchased from MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number: Lot No.: 30842

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 1 hour
- Spectrophotometer: microplate reader (Versamax® Molecular Devices)
The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
- The test item is identified as non-irritant to skin in accordance with UN GHS / EU CLP “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

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
DPBS (MatTek)
- Amount applied: 30 µL

POSITIVE CONTROL
5% SDS solution in deionised water (MatTek)
- Amount applied: 30 µL

Duration of treatment / exposure:

After pre-warming of the EpiDerm™ tissues was completed, the negative and positive control, and the test item was added atop the tissues. The test item respectively controls were tested in triplicate tissues with an exposure time of 60 minutes. Within this period the 6-well plates were placed in the incubator for 34 minutes at standard incubation conditions. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.

Duration of post-treatment incubation (if applicable):

After treatment and washing, the tissues were incubated in 0.9 mL of fresh assay medium for 24 hours. After this incubation period the medium was changed and the tissues were incubated for another about 18 hours at standard incubation conditions. The complete incubation time was about 42 hours.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 runs with 3 replicates each

Value:

2.18

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

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: After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the viability compared with the negative control to 2.91%, thus ensuring the validity of the test system
- Acceptance criteria met for variability between replicate measurements: The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 1: Results after treatment with 2,4,6-Collidine and the controls

Treatment Group	Tissue No.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
		Well 1	Well 2	Well 3
Blank		0.039	0.039	0.039	0.039
Negative control	1	1.680	1.603	1.604	1.629	1.656	96.023	3.6	100.0
	2	1.731	1.763	1.746	1.747		103.149
	3	1.711	1.696	1.718	1.708		100.828
Positive control	1	0.089	0.094	0.092	0.091	0.048	3.147	0.2	2.91
	2	0.087	0.087	0.086	0.087		2.857
	3	0.084	0.084	0.085	0.084		2.714
Test item	1	0.080	0.079	0.079	0.079	0.036	2.406	0.2	2.18
	2	0.072	0.072	0.073	0.072		1.999
	3	0.074	0.075	0.075	0.074		2.124

 

Applicant's summary and conclusion

Interpretation of results:

other: The test substance is irritant or corrosive to skin. Further information is necessary to decide if it is to be classifed into category 1 or 2 for skin irritation/corrosion.

Conclusions:

In this study and under the experimental conditions reported, the test item is irritant or corrosive to skin according to UN GHS and EU CLP regulation.

Executive summary:

The in vitro study according to OECD Guideline No. 439 was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic colour was not intensive and it did not change colour when mixed with deionised water or isopropanol. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item the mean relative viability value was 2.18% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant or corrosive to skin.