Dibutyl [(dipropoxyphosphinothioyl)thio]succinate

Endpoint summary

Description of key information

Skin irritation:

The relative mean tissue viability for the test item, butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester, was 88.2%, greater than 50%, therefore, the test item was classified as non-irritant to skin.

 

The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).

 

The test item was not classified as a skin irritant according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) (Envigo Research Limited, 2018).

Eye irritation:

Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item, butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester (Envigo Research Limited, 2018).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2017 to 16 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Description: Light yellow, clear liquid
- Storage: Room temperature (15-25°C) in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Details on test system:

Test for Direct MTT Reduction:
A test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (−14 to −30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, the MTT reducing test item was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Colour Interference with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.

10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker at room temperature, a visual assessment of the colour was made.

Control samples:

other: Negative Control: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++and Mg+ and Positive Control: Sodium Dodecyl Sulphate

Amount/concentration applied:

10 μL (26.3 μL/cm2)

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, each tissue was rinsed before incubating for 42 hours.

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 Hours post exposure incubation period.

Value:

88.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Direct MTT reduction:

The test item was shown to directly reduce MTT in the direct MTT reduction test.

 

Assessment of Colour Interference with the MTT endpoint:

The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

Test Item, Positive Control Item and Negative Control Item:

The relative mean viability of the test item treated tissues was 88.2% (>50%) after a 15-minute exposure period and 42-hour post-exposure incubation period.

 

Quality Criteria:

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 27.7% relative to the negative control treated tissues and the standard deviation value of the viability was 2.2% (≤18%). The positive control acceptance criteria were therefore satisfied.

 

Mean Viabilities of the EPISKIN™ Tissues:

Mean Viabilities of the EPISKIN™Tissues Treatment	OD570(Mean ± SD)	Relative Tissue Viability (Mean ± SD)
Test Item*	0.753 ± 0.064	88.2% ± 7.5%
DPBS (Negative Control)	0.854 ± 0.047	100% ±5.5%
0.5% SDS (Positive Control)	0.236 ± 0.019	27.7% ± 2.2%

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation and UN GHS Not classified for Irritation (category 3 cannot be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. Tissue viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt in the test item treated tissues relative to the negative controls.

 

Triplicate tissues were treated with the test item for 15 minutes. At the end of the exposure period, each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT; therefore, additional non-viable tissues were incorporated into the testing for correction purposes. The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

At the end of the post exposure incubation period the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 570 nm. Data was presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 88.2%% after the 15 minute exposure period and 42 Hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation and UN GHS Not classified for Irritation (category 3

cannot be determined).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

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

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.

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.

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.

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

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).

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available