1,3-bis(trimethylsilyl)urea

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jul 2019

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

Test guideline 1

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 in 2017

Deviations:

yes

Remarks:

technical proficiency not shown; the positive control was 10% (w/v) Benzalkonium chloride in saline since the laboratory historical control data was established with this chemical

Test guideline 2

Qualifier:

equivalent or similar 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 in 2020

Deviations:

yes

Remarks:

technical proficiency not shown; the positive control was 10% (w/v) Benzalkonium chloride in saline since the laboratory historical control data was established with this chemical.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

adopted in 2010

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006.

Version / remarks:

adopted in 2017

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(trimethylsilyl)urea

EC Number:

242-177-9

EC Name:

1,3-bis(trimethylsilyl)urea

Cas Number:

18297-63-7

Molecular formula:

C7H20N2OSi2

IUPAC Name:

1,3-bis(trimethylsilyl)urea

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, Aschaffenburg, Germany
- Characteristics of donor animals: 14 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin / streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes and directly used in the BCOP test on the same day.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin / streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin)

Test system

Vehicle:

physiological saline

Remarks:

20% (w/v) suspension

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20% (w/v) in saline

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 10% (w/v) in saline

Duration of treatment / exposure:

240 min at 32 ± 1 °C

Number of animals or in vitro replicates:

Three corneas were used for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin). The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Following equilibration, an initial opacity reading was performed to determine the basal opacity (t0). Only corneas with a value of the basal opacity < 7 were used.

NUMBER OF REPLICATES
Sets of three corneas were used for treatment with the test item and for the negative and positive controls, respectively.

TREATMENT METHOD: Open chamber method
Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O- ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed and the basal opacity was determined (t0). For treatment, the anterior compartment received the test item suspension or the negative or positive controls at a volume of 750 µL each on the surface of the corneas. The corneas were incubated in a horizontal position at 32 ± 1 °C in the waterbath for 240 min.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of the test item the corneae were given a final rinse with cMEM without phenol red.

- POST-EXPOSURE INCUBATION: Not performed

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Following exposure, fresh cMEM was added into the anterior compartment and the opacity determinations were performed. Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax® Molecular Devices). The optical density at 490 nm (OD490) of each sampling tube was determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

240 min exposure

Value:

1.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not included by the testing laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses result in opacity (0.33) and permeability values (0.060) that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value (90.93 ± 6.32) which fell within two standard deviations of the current historical mean.

Any other information on results incl. tables

Table 2: Results of the BCOP test after 240 min of exposure

Test Group	Opacity value: Difference (t240 - t0) opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS ± SD	Evaluation
	Individual	Mean	Individual	Mean
Negative Control	1.0	0.33	0.062	0.06	1.93	1.24 ± 0.60	No Category
	0.0		0.060		0.90
	0.0		0.059		0.89
Positive Control	83.67*		0.381*		89.38	90.93 ± 6.32	Category 1
	79.67*		0.391*		85.53
	91.67*		0.415*		97.89
Test item	1.67*		0.00**		1.67	1.04 ± 0.54	No Category
	0.67*		0.00**		0.67
	0.67*		0.009*		0.80

* corrected values

** value was set to zero since the calculated value was negative

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

CLP: Not classified