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

Endpoint summary

Description of key information

Skin irritation/corrosion:

An in vitro study was performed to assess the skin irritation potential of N-tert.-Butoxycarbonyl-4-oxo-L-proline by means of the Human Skin Model Test. The mean relative viability value was 95.08% compared to the relative absorbance value of the negative control. Since this value is above the threshold for irritancy of ≤ 50%, the test item is considered to possess no skin irritation potential. [2019-5124-DGT]

An in vitro study was performed to assess the corrosive potential of N-tert.-Butoxycarbonyl-4-oxo-L-proline by means of the Human Skin Model Test with EpiDerm™ tissues models. After exposure of the tissues to the test item the relative absorbance value was 103.29% after 3 minutes exposure and 92.46% after 1 hour exposure. Both values did not exceed the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be not skin corrosive. [2019-5110-DGT]

Eye irritation/corrosion:

An in vitro study was performed to assess the eye irritation potential of N-tert.-Butoxycarbonyl-4-oxo-L-proline by means of the Human Cornea Model Test. Since the viability of the tissues decreased below 60% (determined value for the test item 4.81%) no prediction on the endpoint eye irritation can be made from this study in isolation. [2019-5152-DGT]

An in vitro study was performed to assess the corneal irritation and damage potential of N-tert.-Butoxycarbonyl-4-oxo-L-proline by means of the BCOP assay using fresh bovine corneae. The calculated mean in vitro irritancy score was 27.83. According to OECD 437 a prediction for corneal irritation and damage potential cannot be made. [2019-5138-DGT]

An in vitro study was performed to further investigate the eye irritation potential of N-tert.-Butoxycarbonyl-4-oxo-L-proline based on the protocol of OECD 492 complemented by the integration of impedance spectroscopy for long term observation of the applied tissue models. Based on the viability data it would suggest that the test substance is not harmful to the eye (no category), since the viability stayed above 60 %. However, based on the TEER 1000Hz measurement an initial irritative effect that reversed over time could be observed, indicating a category 2 substance (serious eye irritation). [2021 -05305 -ENT]

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2019 - 17 January 2020

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)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): 30842
- Quality assurance date: 15.01.2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: several times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 60 minutes
- Spectrophotometer: microplate reader (Versamax® Molecular Devices)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, if the tissue viability after exposure and post-incubation is less or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg (39.7 mg/cm2 according to guideline)

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

17 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main

Value:

95.08

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Treatment with the positive control induced a decrease in the viability compared with the negative control to 2.91%, thus ensuring the validity of the test system.
After treatment with the test item N-tert.-Butoxycarbonyl-4-oxo-L-proline the mean viability value was 95.08% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, N-tert.-Butoxycarbonyl-4-oxo-L-proline is non-irritant to skin according to UN GHS and EU CLP regulation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 December 2019 - 20 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Updated Guideline adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional sub-category 1B and 1C) and non-corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM
- Tissue batch number(s): 30840
- Date of Certificate of Analysis: 18.12.2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.694 ± 0.081
- Barrier function: 5.08 h
- Sterility: sterile

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 μL of deionised water and 25 ± 2mg (39.7 mg/cm2 according to guideline) of the test item were applied onto the surface of duplicate EpiDermTM tissue.

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

92.46

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

103.29

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water and isopropanol did not lead to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show colour interference.

The test item is considered to be non-corrosive to skin:
• since the tissue viability after 3 minutes exposure is greater than 50% and
• the tissue viability after 1 hour exposure is greater than 15%.

The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.890 to 2.020)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is < 15% compared to the negative control (4.24%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 4.4% to 6.7%)

After exposure to the test item N-tert.-Butoxycarbonyl-4-oxo-L-proline the relative absorbance value was 103.29% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 92.46%. Both values did not exceed the threshold for corrosivity which is defined to be <50% after the 3 minutes exposure and <15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, N-tert.-Butoxycarbonyl-4-oxo-L-proline is non corrosive to skin according to EU CLP and UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 December 2019 - 20 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar 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

GLP compliance:

yes (incl. QA statement)

Species:

human

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
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. 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.

- RhCE tissue construct used, including batch number
MatTek EpiOcularTM Tissue, Lot number: 30639, Keratinocyte strain: 1188KW

- Doses of test chemical and control substances used
50 mg test item; 50 μL negative control (deionised water); 50 μL positive control (methyl acetate)

- Temperature of exposure, post-exposure immersion and post-exposure incubation periods
37 ± 1.5°C and 5 ± 0.5% CO2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
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).

- Description of the method used to quantify MTT formazan
MTT-100 kit purchased from MatTek Corporation (Lot No.: 30639).
For the MTT-Assay, tissues were incubated for 180 min in 300 μl MTT solution. Each tissue was extracted with isopropanol within 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.

- Acceptance criteria
The results are acceptable if:
1) The mean negative control OD is > 0.8 and < 2.8.
2) The mean relative viability of the positive control is below 50% of the main negative control viability.
3) The difference of viability between the duplicate tissues of each test group is < 20 percentage points (p.p.) in the same run.

- Positive and negative control means and acceptance ranges based on historical data
Positive control: 27.15% (mean viability), 6.73%—42.54% (range of viabilities), 0.45 (mean absorption), 0.08—0.78 (range of absorbance)
Negative control: 1.67 (mean absorption), 1.02—2.09 (range of absorbance)

Irritation parameter:

other: Tissue viability [%]

Run / experiment:

6 hours

Value:

4.81

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.8 (2.259 and 2.318), thus showing the quality of the tissues. The upper limit of the historical control range was slightly exceeded, but since the OD is within the recommended range (> 0.8 and < 2.8) given by the OECD 492, this minor deviation is judged as irrelevant and does not compromise the validity of this study.

Treatment with the positive control induced a decrease below 50% viability (32.60%) compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20 p.p. (values between 2.61 p.p. and 4.10 p.p.) in the same run (for test item tissues, positive and negative control tissues).

After treatment with the test item a mean relative viability value of 4.81% 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 N-tert.-Butoxycarbonyl-4-oxo-L-proline from this result in isolation and requires additional information for classification purposes.

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 N-tert.-Butoxycarbonyl-4-oxo-L-proline from this result in isolation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2019

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)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% suspension (w/v) in saline

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin). The corneae were isolated on the same day after delivery of the eyes.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NEGATIVE CONTROL USED
Saline (0.9% (w/v) NaCl in deionised water)

POSITIVE CONTROL USED
10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline using sonication

APPLICATION DOSE AND EXPOSURE TIME
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted 240 minutes.

REMOVAL OF TEST SUBSTANCE
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: microplate reader

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG

Irritation parameter:

cornea opacity score

Run / experiment:

main

Value:

27.83

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.94).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 106.87) corresponding to a classification as serious eye damaging (EU CLP/UN GHS (Category 1).
Relative to the negative control, the test item N-tert.-Butoxycarbonyl-4-oxo-L-proline caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 27.83 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Interpretation of results:

other: prediction cannot be made

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (UN GHS) for N-tert.-Butoxycarbonyl-4-oxo-L-proline.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Based on OECD TG 492, complemented by the integration of impedance spectroscopy.

Qualifier:

equivalent or similar 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:

2019

Principles of method if other than guideline:

The in vitro eye irritation test (EIT) is based on the protocol of the OECD test guideline 492 with the in house developed reconstructed cornea model (RCE). The EIT is complemented by the integration of impedance spectroscopy for long term observation of the applied tissue models.

GLP compliance:

no

Specific details on test material used for the study:

Purity: 99.9%

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 h 15 min

Duration of post- treatment incubation (in vitro):

18 +/- 0.25 h

Number of animals or in vitro replicates:

Duplicates

Details on study design:

The RCE are pre-wet with 20 μl of phosphate buffered saline for 30 min. Duplicates of cornea epithelia models, are exposed for 6 h +/- 15 min (via topical application of the test material with 50 mg (at 37°C, 5% CO2 and 95% humidity in an incubator.
Tissue models treated with water (CAS 7732-18-5) are used as negative control or 10% Benzalkonium chloride (CAS 63449-41-2) as positive control.
After exposure the test substances are rinsed through three washing steps at room temperature (RT). An additional post-soak of 25 +/- 2 min (ensures the removal of excess substance by immersing the models in 5 ml Epilife Medium) at RT. Subsequently, viability is measured after post incubation of 18 +/- 0.25 h solids at 37°C, 5% CO2 and 95% humidity.
Transepithelial electrical resistance at 1000 Hz (TEER 1000 Hz) measurements are performed with a customized device. Barrier integrity of tissue models are evaluated via impedance spectroscopy prior to substance application and after the post incubation and is repeated on day 1, 3, 7 and 11 after test material application. In this study the test
protocol for solids was applied. Acceptability range for negative control before treatment: 4000 NC > 600 > Ohm/cm².

Irritation parameter:

percent tissue viability 

Run / experiment:

Sample 1

Value:

103.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent tissue viability 

Run / experiment:

Sample 2

Value:

99.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean

Value:

101.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

TEER value (Transepithelial Electrical Resistance) 

Run / experiment:

Sample 1

Value:

3 046.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

TEER value (Transepithelial Electrical Resistance) 

Run / experiment:

Sample 2

Value:

3 108.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: Mean Percent Barrier Function

Run / experiment:

Mean

Value:

72.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The test substance showed no reduction of viability below 60% with a mean viability of 101% after application, indicating no category substance for eye irritation by viability.
The test substance displayed a decrease in TEER 1000 Hz below 60% with a mean value of 54% after application and a recovery above 50% with a value of 73% at day 7 indicating a category 2 substance by TEER measurement.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the viability data it would suggest that the test substance is not harmful to the eye (no category), since the viability stayed above 60%. However, based on the TEER 1000 Hz measurement an initial irritative effect that reversed over time could be observed, indicating a category 2 substance (serious eye irritation).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The studies for the endpoints skin irritation and skin corrosion performed with N-tert.-Butoxycarbonyl-4-oxo-L-proline showed valid negative results. Therefore, no classification as skin irritant or skin corrosive according to EU CLP and UN GHS is required.

Based on the result obtained in the BCOP assay, no potential of eye corrosion is indicated for the substance N-tert.-Butoxycarbonyl-4-oxo-L-proline. Based on the result obtained in the Human Cornea Model Test no conclusion on the eye irritation potential can be made. In a second eye irritation study according to OECD 492 with a reconstructed cornea model (RCE) it was concluded that based on the viability data it would suggest no category (since the viability stayed above 60%), however, based on the TEER 1000 Hz measurement an initial irritative effect that reversed over time could be observed, indicating a category 2 substance (serious eye irritation).

Taking into account the overall results of the available eye irritation/corrosion tests, the substance N-tert.-Butoxycarbonyl-4-oxo-L-proline is classified as an eye irritant (category 2) according to EU CLP and UN GHS.