1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 March 2022 to 13 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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)

Deviations:

yes

Remarks:

Due to a delay in delivery, the medium (Hanks' balanced salt solution with Ca++ and Mg++ and containing penicillin/streptomycin) used for transport was not available. Therefore, it was substituted with RPMI 1640 (without phenol red) containing Pen/Strep.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane

EC Number:

222-613-4

EC Name:

1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane

Cas Number:

3555-47-3

Molecular formula:

C12H36O4Si5

IUPAC Name:

tetratrimethylsilyl silicate

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST TISSUE:
- Source: Fresh eyes were collected from the slaughterhouse and were transported in RPMI 1640 medium (without phenol red) containing 1% Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.

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
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 minutes incubation with the test substance at 32 ± 1 ºC

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Three corneas were used for the test substance, positive controls and negative controls

Details on study design:

NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : 0.9% physiological saline

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED : 100% ethanol

APPLICATION DOSE AND EXPOSURE TIME : 750 µl for 10 minutes exposure time at 32 ± 1 °C

TREATMENT METHOD: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir Vion Beef B.V., Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in RPMI 1640 medium (without phenol red) containing 1% Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. 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 RPMI 1640 medium. 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 1640 medium (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.

POST-INCUBATION PERIOD: After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. 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 were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 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: One
- POST-EXPOSURE INCUBATION: Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Inadvertently, after final rinse with complete RPMI 1640 medium and subsequent aspiration, the chambers of cornea no. 9 (test item group) were refilled with medium not before but after post-incubation, shortly before the illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The following formula was used to calculate the opacity:
Opacity= Opacity= ((I₀/I ) - b)/a
where a = 0.025 and b = 0.9894 (values a and b are equipment specific variables empirically determined by the manufacturer).
The 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 re-evaluated periodically.
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: The passage of sodium fluorescein dye measured with the aid of Jenway 6405 UV/VIS spectrophotometry (OD490). The OD490 value of a blank cuvette containing medium without fluorescein is measured before setting the spectrophotometer to zero. The blank value is subtracted automatically at each measurement. All measurements were in the linear range of the spectrophotometer (OD490 should be less than 1.500). The final-corrected OD490 of the test article and the positive control were calculated by subtracting the corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – blank OD490) – average of blank-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.

- Others (e.g, pertinent visual observations, histopathology): Each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) calculated using the following formula: IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: Appropriate decision criteria for Test Guideline 437 were used. The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are:

In vitro irritancy score ≤ 3 has a UN GHS 'No Category',
In vitro irritancy score range > 3; ≤ 55 UN GHS 'No prediction can be made',
In vitro irritancy score >55 has a UN GHS 'Category 1'.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value

Value:

0.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

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 (opacity 2.23; permeability 0.065).

Positive controls validity:

valid

Remarks:

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.

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS: The test conditions were adequate and the test system functioned properly.
- 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 (opacity 2.23; permeability 0.065).
- 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.
- Range of historical values if different from the ones specified in the test guideline: See attached results Table 4

Any other information on results incl. tables

See attached background material for results tables.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the Bovine Corneal Opacity and Permeability (BCOP) Study conducted according to OECD Test Guideline 437 and in compliance with GLP, the test item did not induce ocular irritation through either endpoint and resulted in a mean in vitro irritancy score (IVIS) of 0.30 after 10 ± 1 minutes of treatment. Since the IVIS was ≤ 3, the test item is classified into UN GHS 'No Category'.