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

Skin irritation / corrosion

Administrative data

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

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

Version / remarks:

adopted in 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:

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 material

Test material information

Constituent 1

Reference substance name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

EC Number:

947-918-6

Molecular formula:

not applicable, UVCB substance.

IUPAC Name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

Test material form:

solid

In vitro test system

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

Test animals

Species:

other: in vitro system

Test system

Type of coverage:

other: in vitro system

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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%)

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.