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Description of key information

Skin irritation/corrosion: Skin irritant Category 2 (OECD 439/GLP)

Serious eye damage/eye irritation: Eye damage Category 1 (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:
04 June 2018 - 29 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SUQIAN UNITECH CO., LTD; 2018041002
- Purity: 99.29%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was crushed to a fine powder, using a mortar and a pestle.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
other: DPBS
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The test was carried out with the reconstituted three-dimensional human skin model EpiDerm(MatTek). The certificate of analysis from the EpiDerm™ model is presented in Annex 1.
- Tissue batch number(s): Lot No.: 28623
- Production date: 13 June 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 mins and 25 mins at room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 42 ± 2 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (final concentration)
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm
- Linear OD range of spectrophotometer: 1.0-3.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 2.176 ± 0.022, within the acceptable ranges.
- Barrier function: 4.95 hrs, within the acceptable ranges.
- Morphology: appropriate formation of the epidermal barrier, a presence of a function stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile

NUMBER OF REPLICATE TISSUES: In each time interval three tissues were used per test item, three tissues for the positive control and three tissues for negative control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- N. of replicates : two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU)
- Method of calculation used: Non-specific reduction of MTT (NSMTT) was calculated relative to the negative control of living tissues (NK) according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU)
If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.

To check the colouring potential of the test item 25 mg of the test item were 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.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than 50%.
- The test substance is considered to be non-irrating to skin if the viability after 60 minutes exposure is greater than or equal to 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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS solution; MatTek, CAS No.: 151-21-3, Lot No.: 040418SVA).
Duration of treatment / exposure:
60 ± 1 min (37 ± 1 °C for 35 mins and 25 mins at room temperature)
Duration of post-treatment incubation (if applicable):
42 ± 2 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
9.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
positive control
Value:
3.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The 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, NSMTT equalled 0%.
- Colour interference with MTT: The 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, NSC equalled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.806). (Table 3)
- Acceptance criteria met for positive control: The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% – 1.7%).
- Range of historical values if different from the ones specified in the test guideline: Historical control data (n=40) from 2015-2018 was presented and was within guideline standards (Table 4)
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In the in vitro skin irritation test using the reconstructed human epidermal model EpiDerm, 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was irritating.
Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDerm (183804), reconstructed human epidermis tissue was exposed to 25 mg of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) for 60±1 minutes (35 minutes at 37±1°C and 25 minutes at room temperature). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for 42±2 hours. After removal of the test substance, tissues were incubated with MTT for 3 hours incubation. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.806). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% – 1.7%).

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the positive control, 5% SDS, was 3.7 % of the negative control average value. The average viability of tissues treated by the test item, 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine, was 9.4 % of the negative control average value i.e. viability was > 50 %. According to these results, the test substance is irritating.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
26 June 2018 - 14 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SUQIAN UNITECH CO., LTD; 2018041002
- Purity: 99.29%

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


Species:
cattle
Details on test animals or tissues and environmental conditions:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was suspended with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 18095403, expiry date: 02/2021) and afterwards processed with a dispersing machine to give a 20% concentration.
-3 corneas for the test item.
-3 corneas as negative controls treated with physiological saline 0.9% NaCl.
-3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl.
- 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method) of each cornea.
Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32 ± 1 °C
Number of animals or in vitro replicates:
3 corneas for the test item.
3 corneas as negative controls treated with physiological saline 0.9% NaCl.
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
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 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 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS : The eyes were carefully examined for defects and any defective eyes were discarded.

NUMBER OF REPLICATES: 3 corneas for the test item

NEGATIVE CONTROL USED : 3 corneas as negative controls treated with physiological saline 0.9% NaCl

POSITIVE CONTROL USED : 3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). The corneas were incubated for 4 hours ± 5 minutes incubation at 32 ± 1 °C.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: No

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


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The 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.
- Corneal permeability: 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).
The 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. The 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
The 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) = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:The decision criteria as indicated in Table 1 was used.
Irritation parameter:
cornea opacity score
Run / experiment:
test item
Value:
138.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
70.85
Irritation parameter:
fluorescein retention score
Run / experiment:
test item
Value:
0.014
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.134
Irritation parameter:
in vitro irritation score
Run / experiment:
test item
Value:
138.81
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
0.10
Positive controls validity:
valid
Remarks:
87.87
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: All 3 corneas treated with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine showed complete opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The in vitro irritation score obtained with the positive control (87.87) fell within the two standard deviations of the current historical mean (86.15-159.86) and therefore this assay is considered to be valid.
- Range of historical values if different from the ones specified in the test guideline: Historical control data (n=37) was provided from 2015-2018 (Tables 5, 6).
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The following mean in vitro irritation score was 138.81. According to the evaluation criteria the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is classified into UN GHS Category 1.
Executive summary:

In the Bovine Corneal Opacity and Permeability (BCOP) assay (183805), isolated bovine corneas were exposed to 750 µL 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in physiological saline 0.9% NaCl for 4 hours ± 5 minutes incubation at 32 ± 1 °C using the closed chamber method. Physiological saline 0.9% NaCl was used for the negative control and imidazole 20% in physiological saline 0.9% NaCl was used for the positive control. The corneas were rinsed 3 times and then the opacity and permeability (via sodium fluorescein dye) of each cornea were recorded.

The positive control gave the appropriate response. All 3 corneas treated with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine showed complete opacity of the tissue. The mean opacity value for the test substance was 138.67. The mean permeability OD490 for the test substance was 0.014. The IVIS for the test substance was 138.81. The IVIS for 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is > 55 therefore the classification of test substance for eye irritation or serious eye damage is: Category 1.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irriation/corrosion:

There is one in vitro skin irritation test available.

In an in vitro skin irritation assay in a human epidermal model EpiDerm (OECD 439/GLP), reconstructed human epidermis tissue was exposed to 25 mg of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) for 60±1 minutes (35 minutes at 37±1°C and 25 minutes at room temperature). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for 42±2 hours. After removal of the test substance, tissues were incubated with MTT for 3 hours incubation. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.806). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% – 1.7%). The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the positive control, 5% SDS, was 3.7 % of the negative control average value. The average viability of tissues treated by the test item, 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine, was 9.4 % of the negative control average value i.e. viability was > 50 %. According to these results, the test substance is irritating.



Serious eye damage/eye irritation:

There is one in vitro eye irritation test available.

In the Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), isolated bovine corneas were exposed to 750 µL 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in physiological saline 0.9% NaCl for 4 hours ± 5 minutes incubation at 32 ± 1 °C using the closed chamber method. Physiological saline 0.9% NaCl was used for the negative control and imidazole 20% in physiological saline 0.9% NaCl was used for the positive control. The corneas were rinsed 3 times and then the opacity and permeability (via sodium fluorescein dye) of each cornea were recorded. The positive control gave the appropriate response. All 3 corneas treated with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine showed complete opacity of the tissue. The mean opacity value for the test substance was 138.67. The mean permeability OD490 for the test substance was 0.014. The IVIS for the test substance was 138.81. The IVIS for 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine is > 55 therefore the classification of test substance for eye irritation or serious eye damage is: Category 1.

Justification for classification or non-classification

Based on the available information in the dossier, the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (CAS No. 72058-41-4) is classified as Skin irritation Category 2  for skin irritation/corrosion and is classified as Eye damage Category 1 for serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.