Reaction Mass of Distillates (petroleum), hydrotreated heavy paraffinic and Residual oils (petroleum), solvent refined; oxidized, calcium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Deviations:

yes

Remarks:

IVIS score for positive control group higher than expected with no impact on results or integrity of the study (see below)

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)

Deviations:

yes

Remarks:

IVIS score for positive control group higher than expected with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Dark brown extremely viscous liquid
- Storage conditions: Room temperature in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF BOVINE EYES
- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Excised eyes were transported to the test facility over ice packs on the same day of slaughter.
- Corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

120 minutes

Number of animals or in vitro replicates:

Three

Details on study design:

REFERENCE ITEM PREPARATION
- The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.
- The positive control item (ethanol) was used as supplied.

PREPARATION OF CORNEAS
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
- The medium from both chambers of each holder was replaced with fresh complete EMEM.
- A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (see Annex 1, attached). The average opacity for all corneas was calculated.
- Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the cornea to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.
- At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
- The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
- After incubation, the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated.
- The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL).
- The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
- After incubation the medium in the posterior chamber of each holder was decanted and retained.
- Medium representing each cornea (360 μL) was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
- The corneas were retained after testing for possible conduct of histopathology.
- Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface.
- The cassette was immersed in 10% neutral buffered formalin.

DATA EVALUATION
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
- The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading.
- These values were then corrected by subtracting the average change in opacity observed for the negative control corneas.
- The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
- The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea.
- The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

VISUAL OBSERVATION
- The condition of the cornea was visually assessed post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
- Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.
- Sodium chloride solution (0.9% w/v) was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 2.9 and for permeability ≤ 0.103.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

2.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL OPACITY AND PERMEABILITY MEASUREMENTS
- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (attached).

CORNEAL EPITHELIUM CONDITION
- The condition of each cornea is given in Appendix 2 (attached)
- The corneas treated with the test item were clear post-treatment and post-incubation.
- The corneas treated with the negative control item were clear post-treatment and post-incubation.
- The corneas treated with the positive control item were cloudy post-treatment and post-incubation.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 2.1for the test item, 0.8 for the negative control and 52.3 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was marginally above the range of 29.6 to 52.0.
The positive control acceptance criterion was therefore not satisfied and this was reported as a
deviation.
- The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In Vitro Irritancy Scores were reported as 2.1 for the test item, 0.8 for the negative control and 52.3 for the positive control.

Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

METHODS

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

 

RESULTS

In Vitro Irritancy Scores were reported as 2.1 for the test item, 0.8 for the negative control and 52.3 for the positive control.