1,3,5-tris(3-mercaptopropyl)-1,3,5-triazinane-2,4,6-trione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-19 to 2019-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tris(3-sulfanylpropyl)-1,3,5-triazinane-2,4,6-trione

EC Number:

813-556-1

Cas Number:

78366-85-5

Molecular formula:

C12H21N3O3S3

IUPAC Name:

tris(3-sulfanylpropyl)-1,3,5-triazinane-2,4,6-trione

Test material form:

liquid

Details on test material:

- Name: 1,3,5-Tris-(3-mercaptopropyl)isocyanurate
- CAS: 78366-85-5
- Purity: 94.5% (HPLC)
- Batch No: SG0117
- Molecular weight: 351.5 g/mol
- Physical state: liquid
- Colour: colourless
- Storage conditions: room temperature

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as provided by the sponsor

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): no data
- Storage, temperature and transport conditions of ocular tissue: On the test day, 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.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: no data

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

3 corneas each for the test item, negative control and positive control

Details on study design:

SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded. 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 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

TREATMENT METHOD: closed chamber

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The 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). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red).

POST-EXPOSURE INCUBATION:
The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 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 was determined.

METHODS FOR MEASURED ENDPOINTS:
- The optical density was determined at 490 nm (OD490) using a spectrophotometer (Jenway 6405 UV/VIS).

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

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (triplicates)

Value:

23.13

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: According to the UN GHS, this IVIS does not allow to make any prediction

Other effects / acceptance of results:

All 3 corneas treated with 1,3,5-Tris-(3-mercaptopropyl)isocyanurate showed slight opacity of the tissue and were spotted with test item residues. 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 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. For details on the results see box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: In vitro Irritation Score (IVIS)

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.18	0.004	-0.16
2		-0.07	0.005
3		-0.79	0.004
MV		-0.23	0.004
1	Positive Control	35.52	1.438	56.27
2		36.19	1.078
3		31.98	1.826
MV		34.56	1.447
1	Test Item	24.67	0.010	23.13
2		15.97	0.013
3		28.02	0.028
MV		22.88	0.017

MV = mean value

Applicant's summary and conclusion

Interpretation of results:

other: mean in vitro irritation score does not allow to make any prediction

Conclusions:

In conclusion, based on the mean in vitro irritation score of 23.13 obtained in the bovine corneal opacity and permeability assay (OECD 437), no prediction can be made regarding the classification of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate.

Executive summary:

The eye irritation potential of the test substance was investigated in the bovine corneal opacity and permeability assay (OECD 437). 750 µL of the test item (purity: 94.5%) was applied undiluted to the corneas as supplied. A mean in vitro irritation score of 23.13 was determined. 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 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. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate.