Vinylcyclohexane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017- 08-17 to 2018-01-16

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 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 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Vinylcyclohexane

EC Number:

211-779-3

EC Name:

Vinylcyclohexane

Cas Number:

695-12-5

Molecular formula:

C8H14

IUPAC Name:

ethenylcyclohexane

Test material form:

liquid

Details on test material:

- Name: Vinylcyclohexane
- CAS No.: 695-12-5
- Batch No.: VCH/7/17K1
- Expiry date: 30 March 2019
- Purity: 99.8 %
- Appearance: Clear, colourless liquid

Specific details on test material used for the study:

- Storage conditions: Fridge: (2-8 °C), keep container tightly closed, keep away from ignition sources
- Stability: stable under normal temperature and pressure
- Solubility: H2O: < 0,1 g/L ; EtOH: >1 g/L; acetone: >1 g/L; CH3CN: >1 g/L; DMSO: >1 g/L
- Treatment of test material prior to testing: The test item is a liquid substance. It was tested directly, without dilution or preparation of a solution.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Age: 12 to 60 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1 °C
- The eyes were transported to the test facility in Hanks' Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Steptomycin 100 µg/mL) in a suitable cooled container.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

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:

SELECTION AND PREPARATION OF CORNEAS: The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS: After arrival of the corneas, they were examined. Only corneas which were free from damages were used.

NUMBER OF REPLICATES: 3 for each treatment group

NEGATIVE CONTROL USED: HBSS (Hank’s Balanced Salt Solution 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20170831)

POSITIVE CONTROL USED: Dimethylformamide (undiluted)

APPLICATION DOSE AND EXPOSURE TIME: 750 µL application dose, 2 hours exposure time

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 10 min exposure time the corneas were rinsed thoroughly with cMEM without phenol red and afterwards they were finally rinsed also with cMEM without phenol red. Then, the anterior chamber was filled with cMEM without phenol red and the corneas were stored additional 2 hours at 32 ± 1 °C.
- POST-EXPOSURE INCUBATION: 2 hours, afterwards the cMEM was renewed in both chambers (front and anterior chamber). Then, the final opacity value of each cornea was recorded. The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid with a microtiter plate photometer at 492 nm.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity is measured quantitatively as the amount of light transmission through the cornea
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
Opacity = [(I0/1)-b]/a
a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
l0 = the empirically determined illuminance through a cornea holder with windows and
Medium, here: I0= 1093.96
I = the measured illuminance (unit: LUX)
- Corneal permeability: Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber.
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from
the following equation:
IVIS = (opacity difference — mean opacity difference of the negative control) + [15 x (OD492-mean OD492 of the negative control)]
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1 (see "Any other information on materials and methods").
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made. For this purpose, further testing with another suitable method is required.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (triplicates)

Value:

4.48

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, based on the mean in vitro irritation score of 4.48 in the bovine corneal opacity and permeability assay (OECD 437), no prediction can be made on classification, since according to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, cannot be classified in an UN GHS Category.

Executive summary:

In a primary eye irritation study,conducted according to OECD Guideline 437, 750 µL of Vinylcyclohexane (Purity 99.8%) was applied to the bovine corneas of slaughtered cattle 12- 60 months old for 10 minutes at at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.  Irritation was scored by the method of in vitro Irritancy Score. The test substance showed an IVIS of 4.48, which is in the range > 3 and ≤ 55, and therefore, no prediction can be made on classification.