Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

Endpoint summary

Description of key information

Skin irritation/ corrosion (OECD 431 and OECD 439): not irritating

Eye irritation (OECD 437 and OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 - 23 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation 440/2008 of 30 May 2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200), MaTek Corporation, Bratislava, Slovakia

Justification for test system used:

The EpiDerm™ Skin Model is a well-established validated organotypic, three-dimensional model of the human epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST METHOD:
A reconstructed three-dimensional skin model is used based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers. Irritant properties are determined through a decrease in cell viability as determined by a MTT reduction assay.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 25882

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min-exposure); 37 ± 1.5 °C (60 min-exposure)
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure the tissues were removed from the 6-well plate with forceps; using a wash bottle the tissues were gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item; excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: plate spectrophotometer (not further specified)
- Wavelength: 570 nm

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 1.52 ± 0.086 (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 7.27 h (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not show reduction of MTT, therefore no tests with control tissues were performed.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the 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 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 applied: 25 mg; applied directly to the EpiDerm™ tissue using an application spoon avoiding compression of the test item; to ensure good contact with the skin the test item was moistened with 25 µL H2O; the test item was spread to match the size of the tissue.

VEHICLE
- not applicable

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

duplicates for each treatment and control group

Species:

other: in vitro system

Type of coverage:

other: in vitro system

Irritation / corrosion parameter:

% tissue viability

Remarks:

test item

Run / experiment:

3 min

Value:

105

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

test item

Run / experiment:

60 min

Value:

99.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

positive control

Run / experiment:

3 min

Value:

11.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

positive control

Run / experiment:

60 min

Value:

5.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of test item and MTT medium showed no reduction of MTT compared with the solvent.
- Colour interference with MTT: The mixture of test item and distilled water and isopropanol showed no colouring compared with the solvent.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8 (1.769 and 1.900, respectively)
- Acceptance criteria met for positive control: the mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15% (5.1%)
- Acceptance criteria met for variability between replicate measurements: the coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30% (1.6 - 9.1%)

Table 1. Results of 3 min-experiment

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.730	1.801	1.766	1.947	0.193	0.300
	1.702	1.800	1.748	1.949	0.203	0.278
	1.784	1.800	1.769	1.958	0.227	0.287
OD570 - Blank Corrected	1.685	1.759	1.721	1.902	0.148	0.255
	1.657	1.755	1.703	1.904	0.158	0.233
	1.739	1.755	1.724	1.913	0.182	0.242
Mean OD570 of 3 Aliquots (Blank Corrected)	1.694	1.755	1.716	1.906	0.163	0.243
SD OD570 of 3 Aliquots	0.042	0.001	0.011	0.006	0.017	0.011
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.724*		1.811		0.203
SD OD570 of 2 Replicate Tissues	0.043		0.135		0.057
Mean Relative Tissue Viability [%]	100.0		105.5		11.8
Coefficient Of Variation [%]***	2.5		7.4		28.0

*          corrected mean 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 2:  Results of 60 min-experiment

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.763	2.027	1.899	1.879	0.096	0.176
	1.786	2.030	1.922	1.872	0.099	0.182
	1.793	2.003	1.915	1.863	0.102	0.181
OD570 - Blank Corrected	1.736	1.975	1.854	1.834	0.051	0.131
	1.741	1.985	1.877	1.827	0.054	0.137
	1.748	1.958	1.870	1.818	0.057	0.136
Mean OD570 of 3 Aliquots (Blank Corrected)	1.736	1.975	1.867	1.826	0.054	0.135
SD OD570 of 3 Aliquots	0.016	0.015	0.012	0.008	0.003	0.003
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.856*		1.847		0.0594
Mean Relative Tissue Viability [%]	100.0		99.5		5.1**
Coefficient Of Variation [%]***	9.1		1.6		60.3

*          corrected mean 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%.

Table 3. Historical control data

	Mean	SD	n
OD570 of Negative Control (3 min Experiment)	1.895	0.313	10
OD570 of Negative Control (60 min Experiment)	1.867	0.261	11
Relative Tissue Viability [%] of Positive Control (60 min experiment)	6.1	1.99	11
CV [%] (in the range of 20 – 100% viability)	7.8	7.5	31

Historical control data were generated from 2015 - 2016.

Interpretation of results:

other: not corrosive

Conclusions:

Under the conditions of the reconstructed human epidermis test the test substance does not possess corrosive properties. There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat. 1, 1A, 1B/C) based on a positive result in the Reconstructed human epidermis test method (in vitro skin corrosion). A negative in vitro corrosivity response is not conclusive with respect to non-classification or classification as a skin irritant and therefore requires further evaluation and/or data generation.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Mar - 27 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (Epi-200)

Justification for test system used:

The EpiDerm™ Skin Model is a well-established validated organotypic, three-dimensional model of the human epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (Epi-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 25899

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 min in the incubator; 25 ± 1 min at room temperatur in a sterile bench
- Temperature of post-treatment incubation: 37 ± 1 °C for 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm without reference wavelength

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 1.925 ± 0.132 (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 6.52 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.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance showed no color interference 1 h after incubation in deionised water and isopropanol and did not reduce MTT, no additional test was necessary.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 h exposure and is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after 1 h exposure and post-incubation is > 50%.

ACCEPTABILITY OF THE ASSAY
1. The mean OD 570 nm of the negative control tissue is ≥ 0.8 and ≤ 2.8.
2. Mean relative tissue viability of the positive control is ≤ 20%.
3. The relative standard deviation of 3 identical replicates should be ≤ 18%.
OD values should not be below historically established boundaries.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg moistened with 25 mL DPBS

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 tissues

Run / experiment:

60 min

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test item did not show reduction of MTT compared with the solvent.
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water or isopropanol.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.849) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.2%, confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values of the test substance, the positive and negative controls in the main test were 0.4% - 3.6% (threshold of OECD 439: ≤ 18%), showing the validity of the study.

Table 1. Historical control data

	OD570±30 nm Negative control	Relative Viability [%] Positive control	SD Viability [%]
Mean	1.843	4.3	4.2
SD	0.286	2.2	4.7
n	22	22	84

Data of studies performed from 2015 to 2017.

Table 2. Results after treatment with the test item and controls

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.824	1.879	1.802	0.09	0.106	0.099	1.938	1.878	1.825
	1.898	1.893	1.8	0.094	0.105	0.109	1.968	1.878	1.824
OD570(blank corrected)	1.781	1.836	1.759	0.047	0.063	0.056	1.895	1.834	1.782
	1.855	1.849	1.757	0.051	0.062	0.066	1.924	1.835	1.781
Mean OD570 of the duplicates (blank corrected)	1.818	1.843	1.758	0.049	0.063	0.061	1.91	1.835	1.782
Total mean OD570 of 3 replicate tissues (blank corrected)	1.806*			0.058			1.842
SD OD570	0.044			0.007			0.064
Relative tissue viability [%]	100.6	102	97.3	2.7	3.5	3.4	105.7	101.6	98.6
Mean Relative Tissue Viability [%]	100			3.2**			102
SD tissue viability [%]***	2.4			0.4			3.6
CV [% Viabilities]	2.4			12.9			3.5

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

** Mean relative tissue viability of the three positive control tissues is ≤ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Interpretation of results:

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

Conclusions:

Under conditions of the reconstructed human epidermis test, a tissue viability of 102.0% was determined (threshold for classification ≤ 50%). Threfore, the test substance is not considered to possess an irritant potential to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in Hanks´ Balanced Salt Solution (HBSS) containing Pen/strep on ice.
- Time interval prior to initiating testing: The corneae preparation started on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.

Vehicle:

other:

Remarks:

test substance was moistened with physiological saline 0.9% NaCl

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL
- Amount applied: 750 µL
- Concentration: 0.9% NaCl

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20% in physiological saline (0.9% NaCl)

Duration of treatment / exposure:

4 h ± 5 min at 32 ± 2.6 °C

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with Roswell Park Memorial Institute medium (RPMI, without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 2.6 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, an initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.


TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: corneae were washed at least three times with MEM (containing phenol red)


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EHS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

4 h

Value:

0.18

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.74). The positive control (imidazole 20%) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 149.42).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 3. In Vitro Irritancy Scores

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1		0.71	0.024
2	Negative	0.37	0.015
3	Control	0.37	0.012
MV		0.49	0.017	0.74
4		88.86	2.838
5	Positive	116.87	2.198
6	Control	129.66	2.488
MV		111.80	2.508	149.42
7		-0.26	-0.004
8	Test Item	0.88	0.019
9		-0.27	-0.003
MV		0.12	0.004	0.18

MV = mean value

Interpretation of results:

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

Conclusions:

Under conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test subtance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.18

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May - 08 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Species:

human

Strain:

other: EpiOcular™

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years and for a great variety of chemicals.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

The test was performed on a total of 2 tissues per dose group.

Duration of treatment / exposure:

6 ± 0.25 h

Duration of post- treatment incubation (in vitro):

post-exposure immersion: 25 ± 2 min
post-treatment incubation: 18 ± 0.25 h

Details on study design:

- RhCE tissue construct used, including batch number : EpiOcular™ tissues (OCL-200-EIT; MatTek, Lot No.: 27044)

MTT-REDUCING PRE-EXPERIMENT
To check the non-specific MTT-reducing capability of the test item 50 mg of the test item were mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. If the mixture turned blue/purple, the test item was presumed to have reduced MTT. The part of absorption due to the non-specific reduction of MTT was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues was above the 60% threshold value. For quantitative correction of results, two killed tissues were treated with 50 mg of the test item (KT) and one tissue was treated with 50 μl of the negative control (Aqua dest.), respectively. Non-specific reduction of MTT was calculated relative to the negative control of living tissues.

COLOURING POTENTIAL PRE-EXPERIMENT
To check the colouring potential of the test item 50 mg of the test item were mixed per 1 mL Aqua dest. and per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation period, 2 x 200 μL aliquots per test solution were transferred into a 96-well plate, using 200 μL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer. 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 if the mean relative tissue viability of the test item treated tissues was above the 60% threshold value.

EXPERIMENTAL PROCEDURE
On receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 μL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plates were incubated for 6 ± 0.25 h at 37 ± 2 °C, 5.0% CO2 / 95% air. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 ± 2°C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
For each tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

PREDICTION MODEL
The ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. The test item is considered to be irritant to the eye if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant if relative tissue viability is higher than 60%.

ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

Irritation parameter:

other: mean relative viability (%)

Run / experiment:

6 h

Value:

96.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test item showed no reduction of MTT as compared to the solvent.
- Colour interference with MTT: The test item showed no colouring as compared to the solvent.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.796) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (19.1%).
- The difference of rel. absorbance between the two relating tissues of a single item is < 20% (tissue viabilities between 106.7% to 93.3% for negative control, resulting in a relative tissue viability difference of 13.4%; relative tissue viabilities for test item and positive control were 11.1% and 6.8%, respectively).

Table 1: Results of the test item

	Negative control		Positive control		Test item
Tissue	1	2	1	2	1	2
OD570values	1.926	1.679	0.320	0.440	1.839	1.634
	1.900	1.678	0.318	0.437	1.833	1.648
OD570values (blank-corrected)	1.881	1.635	0.276	0.395	1.794	1.589
	1.855	1.634	0.273	0.392	1.789	1.604
Mean of duplicates	1.868	1.634	0.274	0.394	1.792	1.596
Mean OD	1.751*		0.334		1.694
Mean SD OD	0.165		0.084		0.138
Tissue viability [%]	106.7	93.3	15.7	22.5	102.3	91.2
Relative tissue viability difference [%]***	13.4		6.8		11.1
Mean tissue viability [%]	100.0		19.1**		96.7

*: 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%

Table 2: Historical data

	Absolute OD570[nm], negative control	Relative viability [%], positive control	Difference of viability [%]
Mean	1.687	24.6	6.9
SD	0.272	12.3	9.9
n	44	44	178

Historical control data were generated from 2012 – 2018.

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 conducted test, the test substance is not considered to possess an irritating potential towards human cornea in the EpiOcular™ model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion

The skin corrosion potential of Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols has been tested using a reconstructed three-dimensional human epidermis model according to OECD guideline 431 under GLP conditions (Gross, 2018a). The Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. 25 mg of the unchanged test substance was applied to the tissue. In order to ensure good contact with the tissue patch, the test item was moistened with 25 µL water. 50 µL each of the positive (KOH, 8N) and negative control (distilled water) substances were applied to separate tissue patches. The tissue was exposed to test item and control substances for 3 min and 60 min, respectively, and incubated for 3 h post-treatment at 37 ± 1 °C. Experiments were conducted in duplicate for each treatment and control group. After exposure the tissues were gently rinsed about 20 times with phosphate buffered saline (PBS) to remove any residual test or control item. Excess PBS was removed by gently shaking the tissue and blotting the bottom with blotting paper. The mixture of test item and MTT medium showed no reduction of MTT as compared to the solvent. Moreover, the mixture of test item and distilled water and isopropanol showed no coloring as compared to the solvent. Therefore, no tests with control tissues needed to be performed. The mean tissue viability of the test item after 3 min and 60 min exposure was 105% and 99.5%, respectively. The positive control tissue viability was 11.8% and 5.1% after 3 min and 60 min, respectively, showing the experiment was valid. In conclusion, under the conditions of the reconstructed human epidermis test, the test substance did not show corrosive properties. However, no information regarding skin irritation potential or the (non-)classification of the test substance for skin irritation/corrosion can be concluded based on the results of this study.

 

In vitro skin irritation

The skin irritation potential of Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols has been tested using a reconstructed three-dimensional human epidermis model according to OECD guideline 439 under GLP conditions (Gross, 2018b). The Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. 25 mg of the unchanged test substance was applied to the tissue. In order to ensure good contact with the tissue patch, the test item was moistened with 25 µL DPBS. 30 µL each of the positive (SDS, 3%) and negative control (DPBS) substances were applied to separate tissues. The tissue was exposed to test item and control substances for 60 min (25 min at room temperature and 35 min at 37 °C) and incubated for 42 h post-treatment at 37 °C. Experiments were conducted in triplicate for each treatment and control groups. After exposure the tissues were gently rinsed about 20 times with phosphate buffered saline (PBS) to remove any residual test or control item. Excess PBS was removed by gently shaking the tissue and blotting the bottom with blotting paper. The mixture of test item and MTT medium showed no reduction of MTT as compared to the solvent. Moreover, the mixture of test item and distilled water and isopropanol showed no colouring as compared to the solvent. Therefore, no tests with control tissues needed to be performed. The mean tissue viability of the test item after 60 min exposure and 42 h post-incubation was 102.0%. The positive control tissue viability was 3.2%, thus validating the experiment. In conclusion, under the conditions of the reconstructed human epidermis test, the test substance did not show irritating properties.

 

Eye irritation

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability (BCOP) test according to OECD guideline 437 and in compliance with GLP (Niklas, 2018b). After a first opacity measurement of the fresh bovine corneae, 750 µL of the test substance moistened with physiological saline (0.9% NaCl), the negative control (physiological saline; 0.9% (w/v) NaCl) and positive control (imidazole 20% in physiological saline) were applied directly to the epithelial surface of three cattle corneae, respectively, and were incubated in an incubation chamber for 4 h at 32 ± 2.6 °C. After the incubation phase the test substance was rinsed from the corneae and opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation for 90 min at 32 ± 2.6 °C. With the negative control, neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro irritation score (IVIS) of the negative control was 0.74. The positive control, showed clear opacity and distinctive permeability of the corneae fulfilling the criteria for classification as severe irritating/corrosive. The mean IVIS of the positive control was 149.42. All three values of the negative and positive controls, respectively, were within the range of historical control data of the test facility. Relative to the negative control, the test substance caused no increase of the corneal opacity and permeability. The calculated mean IVIS was 0.18. The test substance is not considered to be irritant to the eye.

The potential of the test item to induce eye irritation was also analysed by using the three-dimensional human corneal epithelium model EpiOcular (Gross, 2018c). In this study Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols was applied topically to the EpiOcular tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After a 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay. The ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest. The test item showed no reduction of MTT and no colouring as compared to the solvent. The test item showed no irritant effects since the mean relative tissue viability was > 60%. The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.796). The mean relative tissue viability of the positive control was < 50% and the maximum inter tissue difference of replicate tissues of all dose groups was < 20% (13.4%). In this study the test item showed no irritant effects.

Justification for classification or non-classification

The available data on skin irritation and eye irritation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are, therefore, conclusive but not sufficient for classification.