1,2-Pyrrolidinedicarboxylic acid, 4-hydroxy-, 1-(1,1-dimethylethyl) ester, (2S,4R)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 - 20 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

18 June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model.

Version / remarks:

29 June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2S,4R)-1-[(tert-butoxy)carbonyl]-4-hydroxypyrrolidine-2-carboxylic acid

EC Number:

604-011-7

Cas Number:

13726-69-7

Molecular formula:

C10H17NO5

IUPAC Name:

(2S,4R)-1-[(tert-butoxy)carbonyl]-4-hydroxypyrrolidine-2-carboxylic acid

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg solid test item

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

Pre-experiments:
Two pre-experiments were performed to determine the color interference and the MTT interference.
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08 in the first and did not interfere with MTT in the second pre-experiment, no additional tissues were necessary.

Treatment:
EpiOcularTM tissues were equilibrated at room temperature for 15 min. Then they were conditioned by pre-incubation for 1 hour under standard conditions. After pre-incubation the medium was changed and the tissues were pre-incubated for another approx. 18 h. Prior to application of the test item respectively the controls, all tissues were pre-wetted with 20 μL Ca2+Mg2+free-DPBS and incubated for 30 min.
The test item respectively controls were tested in duplicate tissues.
Concurrent negative and positive control were applied at a volume of 50 μL and for the test item 50 mg to the tissue surface and incubated for 6 h. Afterwards all tissues were rinsed several times and incubated 25 min in 5 ml assay medium at room temperature in a 12-well plate (post exposure immersion). At the end of this incubation the tissues were transferred into a 6-well plate with 1 ml assay medium and were incubated for a post-treatment incubation for 18 h at standard conditions.

MTT-Assay:
Tissues treated with the test item were extracted from the bottom of the tissue only, to minimise any potential contamination of the isopropanol extraction solution with any test item that may have remained on the tissue. The concurrently tested negative and positive control substances were treated similarly to the tested item.
For the MTT-Assay, tissues were incubated for 180 min in 300 μl MTT solution. Each tissue was extracted with isopropanol within about 18 h at 2-8°C without shaking. To mix the extract, the plates were placed on an orbital plate shaker and shaken for approx. 2.5 hours at room temperature. Then, the extracts were mixed and two 200 μL aliquots were transferred to a 96-well plate for OD measurement. 200 μL of isopropanol were added to the wells designated as blanks for 96-well plate.

Measurement:
The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Run / experiment:

1

Value:

2.06

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After treatment with the test item a mean relative viability value of 2.06% was measured compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 60%. Therefore, no prediction can be made for Boc-trans-4-hydroxy-L-proline from this result in isolation and requires additional information for classification purposes.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for Boc-trans-4-hydroxy-L-proline from this result in isolation.