Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation concentrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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:

Update 26/06/2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate

Molecular formula:

Substance is a UVCB and cannot be defined by molecular and structural information

IUPAC Name:

Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate

Test material form:

liquid

Specific details on test material used for the study:

Batch 02 November 2020

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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). They were transported
to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated
on arrival and used within 24 hours of receipt.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume or weight with unit): 0.75 ml neat substance

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also
allocated to the test item and three corneas to the positive control item.

Details on study design:

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v

SOLVENT CONTROL USED (if applicable): no

POSITIVE CONTROL USED: Neat Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 0.75 ml / 10 min

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes. If YES please specify duration: 120 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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
- Corneal permeability: 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS): mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: YES

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

3.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Value 0.3

Positive controls validity:

valid

Remarks:

Value 38.2

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment and post incubation.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control values were within accepted limits.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to UN GHS Classification for the test item Thermal cracking oil from blends of
rubber, fuel oils and paraffin waxes, steam distillation condensate, No stand-alone prediction
of eye irritation can be made under the conditions of the test.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Testing Guideline 437 (Bovine Corneal Opacity and Permeability Test Method). The study was GLPcompliant.
The undiluted test material was applied to three cattle corneas obtained from an abattoir at a volume of 0.75 mL, after which each cornea was incubated at 32±1°C for 10 minutes.

Opacity and permeability of corneas were measured following exposition.
Negative and positive controls were performed and considered as valid.

An In Vitro Irritation Score (IVIS) of 3.8 was calculated for the test item from corneal opacity and permeability measurements. According to UN GHS Classification for the test item Thermal cracking oil from blends of rubber, fuel oils and paraffin waxes, steam distillation condensate, No stand-alone prediction of eye irritation can be made under the conditions of the test.

Since the OECD 437 is designed to identify Eye damage 1 or not classified substances, a decision to classify the substance as eye irritant cat. 2 was taken.