1,3,5-triazine-2,4,6(1H,3H,5H)-trione, zinc salt

Endpoint summary

Description of key information

skin irritation: not irritating (OECD 439, GLP)

eye irritation: not serious eye damaging, but no prediction regarding corneal irritation can be made (OECD 437; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-08 to 2017-08-11

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:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dry in closed container protected from light

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco's phosphate buffered saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (MatTek)
- Tissue lot number: 25835

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min.
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C, 5.0 % CO2 for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature either overnight or, alternatively, at least 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “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(s) applied (volume or weight with unit): 25 mg (39 mg/cm²) of the test item
The test item was applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

82.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture of 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.500).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.9 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of relative tissue viability of replicate tissues of all dose groups was ≤ 18% (0.4 % - 12.3 %).
Please also refer to the field "An other information on results incl. tables" below.

Table 1: Results of the test item 1,3,5 -Triazine-2,4,6(1H,3H,5H)-trione, zinc salt

Name	Negative Control			Positive Control			Test item
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.635	1.509	1.498	0.106	0.096	0.102	1.493	1.146	1.196
	1.595	1.511	1.510	0.110	0.098	0.102	1.487	1.145	1.211
OD570 (blank-corrected)	1.592	1.466	1.455	0.062	0.053	0.059	1.450	1.103	1.153
	1.552	1.468	1.467	0.066	0.055	0.059	1.443	1.101	1.168
mean OD570of the duplicates (blank-corrected)	1.572	1.467	1.461	0.064	0.054	0.059	1.446	1.102	1.160
total mean OD570of 3 replicate tissues (blank-corrected)	1.500*			0.059			1.236
SD OD570	0.062			0.005			0.184
relative tissue viability [%]	104.8	97.8	97.4	4.3	3.6	3.9	96.4	73.5	77.4
mean relative tissue viability [%]	100.0			3.9**			82.4
SD tissue viability [%]***	4.2			0.4			12.3
CV [% viabilities]	4.2			8.9			14.9

^* Blank-corrected mean OD_{570 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%

Table 2: Historical data

	Mean OD570±30nm Negative control (NK)	Mean Relative Viability [%] Positive control (PC)	SD Viability [%]
Mean	1.869	4.1	4.2
SD	0.348	2.0	4.8
n	34	34	121

Historical data were generated from 2009 to 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-23

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:

2013-07-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature; protected from light; dry in closed containers

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals: cattle was between 16 and 60 months old
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Vehicle:

physiological saline

Remarks:

0.9% NaCl

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration: 20% suspension in vehicle

Duration of treatment / exposure:

4 hours ± 5 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (BASF, Duratec) 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 pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- 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 (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 μL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
- after 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
- each cornea was observed visually and pertinent observations were recorded.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation was recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. This I0 value is than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance can be calculated and corneas below this value will be discarded.
- change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
For the IVIS cut-off values for identifying test substances as inducing serious eye damage and test substances not including eye irritation or serious eye damage please refer to table 1 in the field "Any other information onmaterial and methods incl. tables" below.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

14.28

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- all 3 corneas treated with 1,3,5-Triazine-2,4,6(1H,3H,5H)-trione, zinc salt showed opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2017 was used (please refer to tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 were used (please refer to tables in the field "Any other information on results incl. tables" below)

Please also refer for results to the field "Any other information on results incl. tables" below

Table 1: Opacity - Individual data

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	1.98	2.96	0.98
2		2.09	6.01	3.92
3		2.16	3.78	1.62
MV		2.08	4.25	2.17
4	Positive Control	3.50	112.05	108.55	106.38
5		3.54	83.03	79.49	77.32
6		3.74	87.36	83.62	81.45
MV		3.59	94.15	90.55	88.38
7	Test Item	3.23	13.24	10.01	7.84
8		1.01	20.86	19.85	17.68
9		2.20	22.54	20.34	18.17
MV		2.14	18.88	16.73	14.56

MV = mean value

Table 2: Permeability - Individual data

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.019
2		0.022
3		0.028
MV		0.023
4	Positive Control	1.473	1.450
5		2.465	2.442
6		3.065	3.042
MV		2.334	2.311
7	Test Item	0.005	-0.018
8		0.004	-0.019
9		0.004	-0.019
MV		0.004	-0.019

MV = mean value

Table 3: In vitro irritation score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.98	0.019
2		3.92	0.022
3		1.62	0.028
MV		2.17	0.023	2.52
4	Positive Control	106.38	1.450
5		77.32	2.442
6		81.45	3.042
MV		88.38	2.311	123.05
7	Test Item	7.84	-0.018
8		17.68	-0.019
9		18.17	-0.019
MV		14.65	-0.019	14.28

MV = mean value

Table 4: Historial mean In vitro irritation score of the psoitive control from February 2015 until August 2017

	IVIS Positive Control – Imidazole 20%
Mean Value (MV)	125.12
Standard Deviation (SP)	17.16
MV-2xSD	90.81
MV+2xSD	159.44
Number of Replicates providing Historical Mean: 28

Positive controls are updated after every single experiment or at least every 3 months

Table 5: Historical data on opacity and permeability of the positive control (Imidazole 20 %) from August 2017 until October 2017

Incubation;240 min
Number of Replicates providing Historical Mean	Cornea No.	Opacity		Permeability		IVIS
		Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	4	122.785	121.861	0.662	0.624
	5	117.173	116.249	1.220	1.182	133.42
	6	102.655	101.731	2.260	2.222
2	4	108.553	106.381	1.473	1.450
	5	79.491	77.319	2.465	2.442	123.05
	6	83.618	81.446	3.065	3.042
3	4	55.644	56.308	2.200	2.189
	5	71.511	72.175	1.348	1.337	92.54
	6	65.148	65.812	2.040	2.029
Mean Value (MV)		89.620	88.809	1.859	1.835	116.337
Standard Deviation (SP)		23.998	23.370	0.740	0.744	21.251
MV-2xSD		41.624	42.069	0.379	0.347	73.835
MV+2xSD		137.615	135.549	3.340	3.323	158.838

Table 6: Historical mean In vitro irritation score of the negative control from February 2015 until August 2017

	IVIS Negative Control – NaCl 0.9 %
Mean Value (MV)	1.23
Standard Deviation (SP)	0.77
MV-2xSD	-0.31
MV+2xSD	2.78
Number of Replicates providing Historical Mean: 28

Negative controls are updated after every single experiment or at least every three month

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until October 2017

Number of Replicates providing Historical Mean	Cornea No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	0.234	0.008	1.49
	2	1.738	0.008
	3	0.800	0.098
2	1	0.978	0.019	2.52
	2	3.920	0.022
	3	1.617	0.028
3	1	-0.149	0.009	-0.50
	2	-0.415	0.015
	3	-1.427	0.009
1	1	-0.57	0.009	-0.27
	2	-0.11	0.013
	3	-0.53	0.004
2	1	-0.25	0.004	0.66
	2	0.80	0.005
	3	1.17	0.008
Mean Value (MV)		0.520	0.017	0.780
Standard Deviation (SD)		1.296	0.023	1.254
MV-2xSD		-2.072	-0.029	-1.727
MV+2xSD		3.112	0.064	3.287

Interpretation of results:

other: test item is not serious eye damaging (CLP (Cat 1) but the hazardous properties of the test item with regard to corneal irritation cannot be predicted.

Conclusions:

The test substance is not serious eye damaging according to the Regulation (EC) No 1272/2008 and subsequent regulations (Category 1), but no further prediction regarding the eye irritation potential can be made according to the evaluation criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was not observed to be serious eye damaging to the eyes in a reliable in vitro eye irritation study according to OECD 437, but no further prediction regarding the eye irritation potential can be made according to the evaluation criteria.

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess the potential to seriously damage the eyes based on an in vitro OECD 437 (2013) test, but the hazardous properties of the substance with regard to corneal irritation cannot be predicted.