Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Colour: light yellow
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


Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Resultsopen allclose all
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).

Applicant's summary and conclusion

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.