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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 December 2017
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
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:
no
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
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Not available
Cas Number:
2135769-54-7
Molecular formula:
NA
IUPAC Name:
Not available
Test material form:
liquid: viscous
Details on test material:
Vapour pressure: 3.9 x 10-3 Pa
Water solubility: TBC
Density: 1.0
Appearance: black viscous liquid
Melting point: -52 to -21 °C
Boiling point: no boiling and no decomposition up to 300°C
Specific details on test material used for the study:
Identification: Tall Oil Pitch, reaction product with triethylene gl ycol
CAS RN: 2135769-54-7
CAS name: Tall oil pitch, ester with triethylene glycol
Batch: LABO 16-02
Purit y: 100%
Physical state/Appearance: brown viscous liquid
Expiry Date: 14 November 2018
Storage Conditions: room temperature in the dark, under nitrogen

Test animals / tissue source

Species:
other: bovine
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 prepared immediately on arrival

Test system

Vehicle:
not specified
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:
32 ± 1 ºC for 120 minutes
Duration of post- treatment incubation (in vitro):
incubation: 32 ± 1 ºC for 90 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:
SELECTION AND PREPARATION OF CORNEAS
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. The average opacity for all corneas was calculated. 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.


TREATMENT METHOD: The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied 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 ºC 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 3 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.

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 ºC for 90 minutes.

After incubation the medium in the posterior chamber of each holder was decanted and retained. 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.

NEGATIVE CONTROL USED
dentification: Sodium chloride 0.9% w/v
Lot: 3012488
Purit y: 0.9%
Supplier: Aguettant Ltd
Expiry Date: 01 November 2018
Storage Conditions: room temperature

POSITIVE CONTROL USED
Identification: Ethanol
Batch: STBD7546V
Purit y: >99.8%
Supplier: Sigma Aldrich
Expiry Date: 01 April 2018
Storage Conditions: room temperature in the dark

REFERENCE
he negative control item, sodium chloride 0.9% w/v, was used as supplied. The positive control item, ethanol, was used as supplied.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Item
Value:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The
positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control
acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the criteria for classification (No category), the test item Tall Oil Pitch, reaction product with triethylene glycol (CAS: 2135769-54-7) does not require classification to UN GHS or EU CLP.
Executive summary:

The aim of this study was to evaluate the potential of the test item Tall Oil Pitch, reaction product with triethylene glycol to induce serious eye damage. The study was performed according to OECD 437 Guideline (Bovine Corneal Opacity and Permeability (BCOP) test method) and under GLP conditions.

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 anIn Vitro Irritancy Score (IVIS).  

The positive controlIn VitroIrritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of =3.0 and permeability =0.077. The negative control

acceptance criteria were therefore satisfied. 

The In Vitro Irritancy Score of the test item was 1.0.

In conclusion, according to the criteria for classification (No category), the test item Tall Oil Pitch, reaction product with triethylene glycol (CAS: 2135769-54-7) does not require classification to UN GHS or EU CLP.