[(3-Oxo-butyryl)-phenyl-amino]-acetic acid ethyl ester

Endpoint summary

Description of key information

In vitro skin corrosion test in reconstructed human epidermis (OECD 431)

In vitro skin irritation test in reconstructed human epidermis (OECD 439)

Ex vivo eye irritation test (BCOP) (OECD 437)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL NAME (as stated in study report): Intermediate F37

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Valtris Specialty Chemicals / Lot No. W042272
- Expiration date of the lot/batch: November 21, 2019
- Purity: 84.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature In original container as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

Recommended by the OECD and other regulatory authorities

Vehicle:

unchanged (no vehicle)

Details on test system:

Reconstructed human epidermis tissues, SkinEthicTM RHE model, were procured from SkinEthic Laboratories, Episkin 4, Rue Alexander Fleming, 69366 Lyon Cedex 07, France.

After exposure, tissues were rinsed and then dried with cotton buds. Treated tissues were rinsed 20 times in a constant soft stream of 1 mL DPBS at a 5-8 cm distance from the insert to remove all residual test item from the epidermal surface. Mesh (applied on test item treated, negative and positive control tissues) was removed by washing for all tissues. The bottom of tissue inserts were dried on sterile absorbent paper (Kim wipes) for 1-2 seconds. The surface of the stratum corneum was gently swept up using both ends of a cotton tip (5-6 turns per end). After washing, inserts were transferred to holding plates containing 300 µL maintenance medium.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE mode
- Tissue batch number(s): Lot N° 18 RHE 029

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature; 60 min at 37 deg C

REMOVAL OF TEST MATERIAL AND CONTROLS
After exposure, tissues were rinsed and then dried with cotton buds. Treated tissues were rinsed 20 times in a constant soft stream of 1 mL DPBS at a 5-8 cm distance from the insert to remove all residual test item from the epidermal surface. Mesh (applied on test item treated, negative and positive control tissues) was removed by washing for all tissues. The bottom of tissue inserts were dried on sterile absorbent paper (Kim wipes) for 1-2 seconds. The surface of the stratum corneum was gently swept up using both ends of a cotton tip (5-6 turns per end). After washing, inserts were transferred to holding plates containing 300 µL maintenance medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
After rinsing and drying, the MTT test was performed. Tissues were placed in 300 µL of MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37±1 °C in 5±1% CO2 in a 95% humidified incubator. At the end of the MTT test, tissues were observed for MTT reduction.

NUMBER OF REPLICATE TISSUES: three

PREDICTION MODEL / DECISION CRITERIA
per test guideline

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 µL/0.5 cm2
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes at room temperature
60 minutes at 37 ± 1 °C

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure at room temperature

Value:

100.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure at 37 ± 1 °C

Value:

92.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No
- Colour interference with MTT: slight interference

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Remarks:

Not classified by CLP Criteria

Conclusions:

Under guideline (OECD 431) in vitro test conditions, Intermediate F37 was not corrosive to reconstructed human epidermis tissues (SkinEthicTM RHE model).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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 (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL NAME (as stated in study report): Intermediate F-37

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Batch/Lot No. W042272
- Expiration date of the lot/batch: November 21, 2019
- Purity: 84.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature In original container

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Episkin 4, Rue Alexander Fleming, 69366 Lyon Cedex 07, France

Justification for test system used:

Recommended by the OECD and other regulatory authorities

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): Lot N°18 RHE 029

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 deg C

REMOVAL OF TEST MATERIAL AND CONTROLS
After exposure, tissues were rinsed and then dried with cotton buds. The test item was removed by rinsing 25 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert to remove the entire test item from the epidermal surface. Mesh (applied on test item treated tissues, negative and positive control tissues) was removed during washing all tissues. The bottom of tissue inserts were dried on a sterile absorbent paper (Kim wipes) for 1-2 seconds. The surface of the stratum corneum was gently swept up using both ends of a cotton tip (5-6 turns per end). After washing, inserts were transferred to holding plates containing 300 µL maintenance medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Post incubation, the MTT test was performed. Tissues were placed in MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37 ± 1°C in 5 ± 1% CO2 in a 95% humidified incubator. At the end of MTT test, tissues were observed for MTT reduction. All the adapted control tissues were kept in maintenance media (without MTT) and incubated for 180 minutes at 37± 1°C in 5± 1% CO2 in a 95% humidified incubator.

NUMBER OF REPLICATE TISSUES: three

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test is considered to be irritant to skin in accordance with UN GHS Category 2, if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL/0.5 cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL/0.5 cm2


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL/0.5 cm2
- Concentration (if solution): 5% aqueous

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Value:

82.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The Optical density (OD) values for the negative control replicates were between 1.221 to 1.273, against the guideline requirement of ≥ 0.8 and ≤ 3.0 (≥ 1.2 as per SkinEthic SOP). The OD of the blank was between 0.042 to 0.045 which met the guideline requirement of OD < 0.1. The positive control showed a 1.5% cell viability, against the acceptance criteria of <40% for the SkinEthic RHE model, compared to concurrent negative control. Variation between tissue replicates (i.e. CV% value) was 2.43% for the test item group, 0.00% for positive control and 0.81% for negative control against the guideline requirement of ≤ 18%, which demonstrate the efficiency of the test system, SkinEthicTM RHE model.

Interpretation of results:

GHS criteria not met

Remarks:

Not classified by CLP Criteria

Conclusions:

Under guideline (OECD 439) in vitro test conditions, Intermediate F37 was not an irritant to reconstructed human epidermis tissues (SkinEthicTM RHE model).

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:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL NAME (as stated in study report): Internediate F37

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Valtris Specialty Chemicals Ltd. / Batch/Lot No. W042272
- Expiration date of the lot/batch: November 21, 2019
- Purity: 84.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Containers kept tightly closed in a dry, cool and well ventilated place.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System : Isolated Cornea from the eyes of freshly slaughtered cattle
Source : Deonar Abattoir slaughter house, Mumbai, Maharashtra
Bovine Age : Between 1 to 5 years (age of the animals was determined based on the teeth count and horn ring count along with Horizontal Diameter of Corneas and Central Corneal Thickness)
Transportation Condition : Transported (in a sealed plastic container) under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

approximately 10 minutes ± 30 seconds

Duration of post- treatment incubation (in vitro):

approximately 2 hours ± 10 minutes

Number of animals or in vitro replicates:

three corneas per test group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were used within 24 hours from slaughter. Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.

Corneas, free from defects, were dissected to a 2 to 3 mm rim and were transferred to container containing Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL].

Selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on the top of the cornea and fixed in place. Both chambers were then filled to excess with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) (posterior chamber first to return the cornea to its natural concave position), ensuring no bubbles were present within the holders. The device was then equilibrated at 32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, the medium was removed from both the chambers and fresh pre-warmed phenol red free EMEM was added to both chambers and baseline opacity readings were taken for each cornea.

REMOVAL OF TEST SUBSTANCE
At the end of exposure period, the test item, positive control and negative control were removed from their respective anterior chamber and the corneal epithelium washed until no visual evidence of test item, positive control or negative control was observed using EMEM (containing phenol red).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: light transmission
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: per test guideline

Irritation parameter:

in vitro irritation score

Remarks:

IVIS

Value:

0.53

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

GHS criteria not met

Remarks:

Not classified by CLP Criteria

Conclusions:

Under guideline (OECD 437) test conditions, the mean IVIS score for Intermediate F37 in the bovine corneal opacity and permeabilty (BCOP) test was 0.53. Based on these results, Intermediate F37 is not classified for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on the available data, Intermediate F37 is not classsifed for skin corrosion / irritation or for serious eye damage / eye irritation according to Regulation (EC) No 1272/2008