O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2017 to 08 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

EC Number:

265-967-5

EC Name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Cas Number:

65894-76-0

Molecular formula:

C12H13NO4

IUPAC Name:

O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off white powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue lot number: 25817
- Delivery date: 06 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ° C
- Temperature of post-treatment incubation: 37 °C

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT: 25 mg of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 25 mg of test material was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

MAIN TEST
- The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.
- Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test material and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test material to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.
- When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure.

REMOVAL OF TEST MATERIAL AND CONTROLS AND MTT LOADING
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
- After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

NUMBER OF REPLICATE TISSUES: 2

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

QUANTITATIVE MTT ASSESSMENT
- The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60 Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material/ mean OD570 of the negative control) x 100
Classification of corrosivity potential is based on relative viabilities for both exposure times according to:
- Relative mean tissue viability (% of negative control): 3 min < 50: H314 Category 1A
- Relative mean tissue viability (% of negative control): 3 min ≥ 50; 1hour < 15: H314 Category 1B or 1C
- Relative mean tissue viability (% of negative control): 3 min ≥ 50; 1 hour ≥ 15: Not classified for corrosivity

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control: Potassium Hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15 %.
Coefficient of Variation: In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2 per exposure time

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

99.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.0 % viability

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure period

Value:

98.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

5.1 % viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH MTT
- The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

QUALITY CRITERIA
- The mean OD570 for the negative control treated tissues was 1.494 for the 3 Minute exposure period and 1.501 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 5.1 % relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of Results

Exposure Period	Percentage viability
	Negative control	Positive control	Test material
3 minutes	100*	4.0	99.7
60 minutes	100*	5.1	98.7

 

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

The ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was investigated. The possible corrosive potential was tested through topical application for 3 minutes and 1 hour.

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

The positive control had a mean relative tissue viability of 5.1 % after the 1-hour exposure.

The test material had a mean relative tissue viability of 98.7 % after the 1-hour exposure.

The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.