Vinyltoluene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2020 - 03 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methylstyrene

EC Number:

202-889-2

EC Name:

3-methylstyrene

Cas Number:

100-80-1

Molecular formula:

C9H10

IUPAC Name:

1-methyl-3-vinylbenzene

Constituent 2

Reference substance name:

4-methylstyrene

EC Number:

210-762-8

EC Name:

4-methylstyrene

Cas Number:

622-97-9

Molecular formula:

C9H10

IUPAC Name:

1-methyl-4-vinylbenzene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Batch no.: 20200608
- Expiration: 07 June 2021
- Purity, including information on contaminants, isomers, etc.: 99.66% (3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at +2 °C to +8 °C in a tightly closed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none, the test item was used as supplied.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from cattle were obtained from a slaughterhouse (Otto Vollertsen GmbH, 24986 Mittelangeln, Germany)
- Characteristics of donor animals (e.g. age, sex, weight): 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):To minimise deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks' Balanced Salt Solution 3 (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL
- Selection and preparation of corneas: The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period had to be discarded.
- Quality check of the isolated corneas: Yes

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 (undiluted); closed-chamber method.


VEHICLE
none

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9 % NaCl solution

SOLVENT CONTROL USED (if applicable): none

POSITIVE CONTROL USED: 1 % NaOH solution in highly purified water

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least two times until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber.

- POST-EXPOSURE INCUBATION: The holder was incubated in a horizontal position at 32 ± 1 °C for 2 hrs.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with an opacitometer (BASF 2011-13; 67063 Ludwigshafen am Rhein, Germany) resulting in opacity values measured on a continuous scale.

- Corneal permeability: 1 mL sodium fluorescein solution (4 mg/mL in 0.9 % sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Infinite M200 Pro12). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 based upon a 96-well microtiter plate reader (Tecan Infinite M200 Pro).

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

DECISION CRITERIA: IVIS ≤ 3 : No Category; IVIS> 3 and ≤ 55 : No prediction can be made ; IVIS > 55: Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

1.992

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1.992 ±0.222

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

7.927

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 7.927±2.006

Results 3

Irritation parameter:

fluorescein leakage

Run / experiment:

1

Value:

0.396

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 0.396 ±0.134

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The corneas treated with the negative control item 0.9 % NaCl solution revealed a mean opacity value of 0.478 ± 1.679 and a mean permeability value of 0.009 ± 0.004. The calculated IVIS value of 0.613 ± 1.731 was well below the cut-off value of 3 (UN GHS no category). One value (2.231) of the triple determination of opacity values of the negative control was slightly higher than the upper limit of acceptance value of the historical background data (1.604). As this value is only slightly higher than the upper limit of acceptance of the historical background data and hence, is not an extreme outlier, this value is acceptable for inclusion into the historical background data. Additionally, the mean opacity value of the negative control is lower than the upper limit of acceptance of the historical control (Appendix 2).

- Acceptance criteria met for positive control: The corneas treated with the positive control item 1 % NaOH in highly purified water solution revealed a mean opacity value of 31.634 ± 10.847 and a mean permeability value of 3.024 ± 1.363 compared to the solvent control. The calculated IVIS value of 76.999 ± 10.459 was within two standard deviations of the current historical mean (Appendix 2) and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In the Bovine Corneal Opacity and Permeability (BCOP) assay, the IVIS value for Vinyltoluene is 7.927 ± 2.006 (> 3 and ≤ 55), therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

Executive summary:

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), 3 isolated bovine corneas were exposed to Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) in liquid form for 10 minutes using the closed-chamber method. 0.9% sodium chloride was used for the negative control and 1% NaOH solution in highly purified water was used for the positive control. The corneas were rinsed and following a 2-hour post-exposure incubation at 32°C ± 1°C, the opacity and permeability of each cornea was recorded.

 

The positive and negative controls gave the appropriate response (IVIS = 76.999 ± 10.459 and 0.613 ± 1.731, respectively). The mean opacity value for the test substance was 1.992 ± 0.222. The mean permeability OD490 for the test substance was 0.396 ± 0.134. The IVIS for the test substance was 7.927 ± 2.006. The IVIS for Vinyltoluene is > 3 and simultaneously ≤ 55, therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.