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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in the Bovine Corneal Opacity and Permeability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 guidelineopen allclose all
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)
Version / remarks:
adopted 09. Oct. 2017: “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:
not specified
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 14. Feb. 2017
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: tert-butylbenzene
- IUPAC name: tert-Butylbenzene
- Molecular formula: C10H14
- Molecular weight: 134.22 g/mol
- Substance type: Organic
- Physical state: Liquid

Test animals / tissue source

Species:
other: Bovine
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Test item: 750 µL
Positive control: 750 µL
Negative control: 750 µL

- Concentration (if solution):
Test item: Tested directly, without dilution or preparation of a solution
Positive control: Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted, batch no.: 475235719
Negative control: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in de-min. water (1:10), batch no.: 20180809
Duration of treatment / exposure:
10 minutes at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 hours at 32 ± 1 °C
Number of animals or in vitro replicates:
3
Details on study design:
-SELECTION AND PREPARATION OF CORNEAS:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

-QUALITY CHECK OF THE ISOLATED CORNEAS:
The corneas were examined and only corneas which were free from damages were used

-NUMBER OF REPLICATES: 3

-NEGATIVE CONTROL USED: Yes (HBSS)

-POSITIVE CONTROL USED: Yes (Dimethylformamide (undiluted))

-APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 10min

-TREATMENT METHOD: Closed Chamber Method

-POST-INCUBATION PERIOD: Yes (2 hours at 32 ± 1 °C)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
Rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red.
- POST-EXPOSURE INCUBATION:
90 minutes at 32 ± 1 °C.

-METHODS FOR MEASURED ENDPOINTS:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

-SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
:
The IVIS of each replicate of the negative control: IVIS = opacity difference + (15 x corrected OD492 value).
The IVIS of each replicate of the positive control and of the test item: IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)].

-DECISION CRITERIA: According to OECD Guideline no. 437 (Oct. 2017).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
29.94
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made.
Executive summary:

This in vitro study was performed to assess corneal damage potential of tert-Butylbenzene by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item tert-Butylbenzene was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

 

The test item was tested neat.

Under the conditions of this test, the test item tert-Butylbenzene showed effects on the cornea of the bovine eye. The calculated mean IVIS was 29.94.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.

 

The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.

 

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.