6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May - 19 Sep 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:

2017

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)

Version / remarks:

adopted Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit, Schwabach, Deutschland

Test material

Test material information

Constituent 1

Reference substance name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

EC Number:

254-074-6

EC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Cas Number:

38668-46-1

Molecular formula:

C9H13N7

IUPAC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200, MatTek, Bratislava, Slovakia)
- Tissue batch number: 25832
- Delivery date: 26 July 2016
- Date of initiation of testing: 26 July 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure), 37 ± 1 °C (60 ± 5 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed using a wash bottle containing phosphate buffered saline (PBS) to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.466 ± 0.03 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.06 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was observed.

NUMBER OF REPLICATE TISSUES: 2 replicates for each treatment condition (3 min and 60 min experiment)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT and showed no colouring as compared to the solvent, an additional test with freeze-killed or viable tissues was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater or equal than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg test substance + 25 µL water (test substance moistened with water to ensure the contact with the skin model)

NEGATIVE CONTROL
- Amount(s) applied (volume): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume): 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

in duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

102.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

98.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water and isopropanol and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time thereby confirming the acceptable quality of the tissues.

- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (14.8%) and for the 60 min exposure period (8.7%) thus confirming the validity of the test system and the specific batch of tissue models (acceptance criteria: mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%)

- Acceptance criteria met for variability between replicate measurements: The coefficient of variation in the range 20 - 100% viability between tissue replicates was ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

Any other information on results incl. tables

Table 2: Results of 3 min Experiment

Name	Negative Control		Test ltem		Positive Control
Tissue	1	2	1	2	1	2
	1.244	1.301	1.253	1. 254	0.188	0.261
Absolute OD570	1.244	1.311	1.346	1.315	0.197	0.261
	1.260	1.345	1.399	1.327	0.198	0.274
	1.198	1.255	1.206	1.207	0.142	0.215
OD570 - Blank Corrected	1.197	1.264	1.299	1.268	0.150	0.214
	1.214	1.299	1.353	1.280	0.152	0.228
Mean OD570 of 3 Aliquots (blank corrected)	1.203	1.273	1.286	1.252	0. 148	0.219
SD OD570 of 3 Aliquots	0.010	0.033	0.071	0.043	0.026	0.026
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.238*		1.269		0.184
SD OD570 of 2 Replicate Tissues	0.049		0.024		0.050
Mean Relative Tissue Viability[%]	100.0		102.5		14.8
Coefficient of Variation[%] **	4.0		1.9		27.3

* corrected mean of OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

Table 3: Results of 60 min Experiment

Name	Negative Control		Test ltem		Positive Control
Tissue	1	2	1	2	1	2
	1.390	1.420	1.360	1.376	0.154	0.172
Absolute OD570	1.439	1.429	1.426	1.382	0.159	0.179
	1.431	1.423	1.454	1.419	0.157	0.175
	1.344	1.374	1.313	1.330	0.108	0.125
OD570 - Blank Corrected	1.393	1.383	1.380	1.336	0.113	0.133
	1.385	1.377	1.408	1.372	0.110	0.128
Mean OD570 of 3 Aliquots (blank corrected)	1.374	1.378	1.367	1.346	0.110	0.129
SD OD570 of 3 Aliquots	0.026	0.026	0.050	0.033	0.025	0.026
Total Mean OD 570 of 2 Replicate Tissues (Blank Corrected)	1.376*		1.356		0.120
SD OD570 of 2 Replicate Tissues	0.003		0.015		0.013
Mean Relative Tissue Viability [%]	100.0		98.6		8.7**
Coefficient Of Variation [%]***	0.2		1.1		10.9

* corrected mean of OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model.
CLP: non-corrosive