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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2017 - 10 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 9 Oct 2017

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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- in vitro test method: Eye Irritation Test (EIT) with reconstructed human cornea-like epithelium (RhCE) tissue, EpiOcular™ (MatTek)
- Lot number: 27015
- Keratinocyte strain: 4F1188
- no biological contaminants (virus, bacteria, yeast or fungi) detected
- Acceptance criteria for tissue viability met: 1.958 ± 0.065 (Acceptance criteria: OD (540-570 nm) [1.1 - 3.0])
- Acceptance criteria for Barrier function met: 25.64 min (Acceptance criteria: ET-50 [12.2 - 37.5])

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: Sigma, RNBF7110

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: Merck, S6943111

Duration of treatment / exposure:

6 ± 0.25 h

Duration of post- treatment incubation (in vitro):

25 ± 2 min (post-soak plate)
18 ± 0.25 h (post-treatment plate)

Number of animals or in vitro replicates:

duplicates for each treatment and control group; from each tissue, 2 absorbance measurements after MTT incubation were performed

Details on study design:

- RhCE tissue construct used, including batch number, Test Kit name: in vitro test method OCL-200-EIT (Epiocular™, MatTek), Lot number: 27015

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure: 6 ± 0.25 h at 37 ± 1°C, post exposure: 25 ± 2 min at room temperature (post-soak plate), 18 ± 0.25 h (post-treatment plate) at 37 ± 1°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to change the color of the MTT medium was assessed in a pre-experiment. Since the MTT solution colour did not change, the test substance is not presumed to have reduced the MTT. To check the colouring potential of the test substance, the test substance were mixed per Aqua dest. and per isopropanol. After an incubation period OD was measured at 570 nm.
- If one of the two ODnet was > 0.08, or if the intrinsic colour of the test item is blue, black or dark-purple, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 50 mg of the test item (TVT) if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. The non-specific colour (NSCliving) was then calculated according to the following formula: NSCliving [%] = [ODTVT(viable test substance)/ODNK]*100
- If the viability difference of the two identically treated additional viable test substance treated tissues (TVT) was > 20% the colour control was considered as non-qualified.
- If NSCliving was ≤ 60% relative to the negative control of living tissues the mean relative tissue viability of the test substance treated tissues (TM) was corrected to the NSC-corrected mean relative tissue viability (NSCCV) which was considered for classification of the test item according to the following formula: NSCCV [%] = viabilityTM [%] – NSCliving [%]
- If NSCliving was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.
- If uncorrected ODTest substance of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.
- If NSCliving was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.
- If uncorrected ODTest substance of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.
- For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 50 mg of the test item (TKT).
- The non-specific colour of additional killed tissues (NSCkilled) was then calculated according to the following formula: NSCkilled [%] = [ODTKT/ODNK]*100
- The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

- Mixture of the test substance per Aqua dest. and per 2 isopropanol showed colouring as compared to the solvent.
Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.
NSCliving [%] = [ODTVT/ODNK]*100
Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.
NSC1 [%] = [ODTVT1 /ODNK] * 100 = [0.0035 / 1.776] * 100 = 0.19%
NSC2 [%] = [ODTVT2 /ODNK] * 100 = [0.0054 / 1.776] * 100 = 0.30%
Mean NSCliving = 0.25%
NSC1 – NSC2 = ±0.11%
NSCliving was ≤ 60% (0.25%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%] = 94.1% - 0.25% = 93.9%

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength: 570 nm ± 30 nm

- Spectrophotometer: plate spectrophotometer

- Description of evaluation criteria used: The test substance is considered to be not irritating to eye if the tissue viability is > 60%. The test substance is considered to be irritating to eye if the tissue viability is ≤ 60%.

- Positive and negative control means and acceptance ranges: The test meets acceptance criteria if: mean absolute OD570 of the negative control is > 0.8 and < 2.5, mean relative tissue viability of the positive control is < 50%
- Acceptable variability between tissue replicates for positive and negative controls and for the test substance: Differences of two tissue cell viabilities in each treatment group are < 20%

Results and discussion

In vitro

Results

Irritation parameter:

other: % non specific color (NSC) corrected mean relative tissue viability (NSCCV) of 2 tissues

Value:

93.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The pre-experiment showed no reduction of MTT by the test substance as compared to the solvent. The mixture did not turn blue/purple.

The pre-experiment to test the colouring potential of the test substance, showed colouring when the test substance was mixed with Aqua dest. or isopropanol as compared to the solvent.
Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.
NSCliving [%] = [ODTVT/ODNK]*100
Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.
NSC1 [%] = [ODTVT1 /ODNK] * 100 = [0.0035 / 1.776] * 100 = 0.19%
NSC2 [%] = [ODTVT2 /ODNK] * 100 = [0.0054 / 1.776] * 100 = 0.30%
Mean NSCliving = 0.25%
NSC1 – NSC2 = ±0.11%
NSCliving was ≤ 60% (0.25%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%] = 94.1% - 0.25% = 93.9%

NSCliving = non-specific colour of additional viable tissue
NSCkilled = non-specific colour of additional killed tissue
OD = optical density
NK = Negative control of living tissue
NSCCV = non-specific colour corrected mean relative tissue viability
TVT = additional test item treated killed tissue without MTT
TM = test item treated living tissues

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD of the negative control was 1.818, 1.847, 1.737 and 1.704, and thus within the acceptibility range (OD > 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes, the mean positive control reduced the cell viability at 19.1% and fulfilled the acceptance criteria (< 50%).
- Acceptance criteria for variabilties: Differences of two tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 6.3%, 2.3% and 18.5%, respectively, and thus < 20%.

Any other information on results incl. tables

Table 1: Summary of Results

Name	Negative Control		Positive Control		Test Substance
Tissue	1	2	1	2	1	2
OD570values (blank-corrected)	1.818	1.737	0.312	0.365	1.493	1.813
	1.847	1.704	0.328	0.356	1.520	1.859
Mean of the duplicates	1.833	1.720	0.320	0.360	1.507	1.836
Mean OD	1.776*		0.340		1.671
Mean SD OD	0.07		0.02		0.19
Tissue viability [%]	103.2	96.8	18.0	20.3	84.8	103.3
Relative tissue viability difference [%]^***	6.3		2.3		18.5
Mean tissue viability [%]	100.0		19.1**		94.1
NSCCV (non-specific clour corrected mean relative tissue viability)	100.0		19.1**		93.9

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

** Mean relative tissue viability of the positive control is < 50%

*** Relative tissue viability difference of replicate tissues is < 20%.

SD = standard deviation

OD = optical density

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of the RhCE test method the test substance did not show irritant properties.
CLP: not classified