Dibutyl [(dipropoxyphosphinothioyl)thio]succinate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 2017 to 26 September 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 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:

no

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:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Cas Number:

68413-47-8

Molecular formula:

C18H35O6PS2

IUPAC Name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Test material form:

liquid

Specific details on test material used for the study:

- Description: Light yellow, clear liquid
- Storage: Room temperature in the dark

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 prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: Sodium chloride 0.9% w/v; Positive control: Ethanol

Amount / concentration applied:

0.75 mL of the test item was applied to the corneas.

Duration of treatment / exposure:

The undiluted test item, butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester, was applied for 10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes.

Number of animals or in vitro replicates:

Three.

Details on study design:

Selection of Corneas and Opacity Reading:
The medium from both chambers of each holder was replaced with fresh complete EMEM.
The corneas were observed for any defects. A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer (Annex 2). 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

Treatment of Corneas:
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.

The negative and positive control data was shared with Envigo - study numbers DQ51LG and FT13LM.

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 ºC for 90 minutes.

Permeability Determinations:
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.

Histopathology:
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. No histopathology was required for this study.

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.

Results and discussion

In vitro

Results

Irritation parameter:

cornea opacity score

Value:

40.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The corneas treated with the test item were opaque post treatment and cloudy 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.

Controls:
The positive control In Vitro Irritancy Score was within the historical 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.

Any other information on results incl. tables

In VitroIrritancy Score

The In Vitroirritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item*	40.5
0.9% (w/v) Sodium Chloride (Negative Control)	0.8
Neat Ethanol (Positive Control)	43.1

Note: * Butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the BCOP test, no prediction of eye irritation can be made for the test substance (Envigo, 2018).