Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9/3/18 - 30/5/18

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Cas Number:

2125692-22-8

Molecular formula:

C25-31H54-66NO5S

IUPAC Name:

Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Test material form:

solid

Test animals / tissue source

Species:

human

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

50mg

Duration of treatment / exposure:

6 hour exposure

Duration of post- treatment incubation (in vitro):

6 hours incubation

Number of animals or in vitro replicates:

2

Details on study design:

The test article may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement.
Therefore, its ability to directly reduce MTT was evaluated.

For this purpose, a 50 mg scoop was used to deliver Aminosensyl to 1 mL of 1 mg/mL MTT solution in a 6-well plate and incubated for 3 hours in the dark at 37° ± 1° C and a humidified atmosphere of 5% 002 ± 1% (SCC). A negative control (50 pL of sterile deionized water) was run concurrently.

Following 3 hour incubation of Aminosensyl with MTT dye solution, the MTT solution remained the same color as the negative control sample to which no test article was added. Therefore, the test article does not reduce MTT and will not interfere with the assay.

OCL-200 tissues were removed from packaging, placed each into one well of a 6-well plate containing 1 mL of pre-warmed (to 37°C) Assay Medium (OCL-200-ASY) and equilibrated at SOC for 1 hour.

After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular tissues were incubated at SCC overnight (17 hours).

Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with 20 iL of Ca++Mg++Free-DPBS and incubated at SOC for 30 minutes.

Exposure: after the pre-treatment, 50 iL of the negative control and positive control and a 50 mg scoop of Aminosensyl was used to deliver topically the NC, PC, and the test article (Aminosensyl) on two (n=2) tissues. Actual weight of the applied material is recorded in the study file.

The tissues were incubated at SCO for 6 hours.

Rinsing: at the end of the treatment, the test articles were removed by rinsing the tissues with 100 mL of Ca/Mg-Free DPBS three times.

Post-Soak: after rinsing, the tissues were immediately transferred to and immersed in 5 mL of Assay Medium (room temperature) for 25 minutes to remove any test article absorbed into the tissue.

Post-incubation: at the end of the Post-Soak immersion, each insert was removed from the Assay Medium. The medium was decanted off the tissue and the insert was blotted on absorbent material and transferred to the 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 18 hours at SCC.

Cytotoxicity Assessment of OCL-200 Tissues via MTT
Just prior to the end of the post-incubation, 2 mL of MTT concentrate (supplied by MatTek, part number MTT-1 00-CON) was added to 8 mL of MTT diluent (supplied by MatTek, part number MTT-100-DIL) to prepare the MTT reagent. The reconstituted MTT reagent was protected from light by covering the tube with aluminum foil. 2. 300 pL of the MTT reagent was added into the appropriate number of wells of a 24-well plate and equilibrated to 37°C.

At the end of the Post-treatment Incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution and incubated for 3 hours at SCC.

At the end of the incubation, the tissues were removed, blotted dry on an absorbent material and moved to a clean 24-well plate containing 2 mL of extractant solution (supplied by MatTek, part number MTT-100-EXT) and extracted overnight at room temperature. The plate was protected from light exposure and sealed to prevent extractant evaporation.

At the end of the extraction period, the extractant solution from the apical compartment was combined with that in the well below, the tissue inserts were removed and discarded.

Extractant solution was mixed and two 200 pL aliquots of each sample were added to a 96-well plate. The optical density of the extracted samples was measured using a spectrophotometer at 570 nm using 200 [iL of extractant solution as a blank.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: viability tissue

Run / experiment:

1

Value:

93.36

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: viability tissue

Run / experiment:

2

Value:

85.27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

All acceptance criteria for the EpiOcular-EIT assay were met.
As per the EpiOcular-EIT prediction model, materials that result in viability >60% are labeled as non-irritants while materials that result in tissue viability 60% are labeled as irritants.
Based on the above categorization TA (Aminosensyl) is categorized as Non-Irritating (NI).

Executive summary:

Study #049-18 is intended to evaluate the ocular irritation potential of topically applied Aminosensyl using the ECVAM validated EpiOcular Eye Irritation Test (EIT) (incorporated into OECD TG 492).

EpiOcular (OCL-200), produced by MatTek Corporation, was used to evaluate ocular irritation

potential of a topically applied test article. Tissue viability following topical application of test article was evaluated via MTT assay.

The EpiOcular-EIT results using EpiOcular tissues from lots #27422 and 27424 are listed in

Tables 3, 4A, and 4B, and Figures 1 and 2.

Since recording for the raw data for first test (“test 1”) incubations couldn’t be verified, re-testing of the test article (Aminosensyl) was performed (“re-test”).

All acceptance criteria for the EpiOcular-EIT assay were met in both test 1 and the re-test

As per the EpiOcular-EIT prediction model, materials that result in viability >60% are labeled as non-irritants while materials that result in tissue viability 60% are labeled as irritants.

The average tissue viability with test article (Aminosensyl) was 93.36% in test 1 and 8