Sebacohydrazide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June - 29 June 2018

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142.

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sebacohydrazide

EC Number:

213-126-8

EC Name:

Sebacohydrazide

Cas Number:

925-83-7

Molecular formula:

C10H22N4O2

IUPAC Name:

sebacohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Test item name (as stated in report): SDH
Test item storage: at room temperature protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

All cells used to produce EpiDermTM are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.
Cells are screened for potential biological contaminants: HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and other fungi. No biological contaminants were detected.

Justification for test system used:

Recommended test system in OECD and EC guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Test system: EpiDerm Skin Model (EPI-200, Lot no.: 28811, kit L and M).
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
-Media used: DMEM (Dulbecco’s Modified Eagle’s Medium) supplied by MatTek Corporation.

ENVIRONMENTAL CONDITIONS
- Test item incubation was carried out for 3 minutes at room temperature
- All other incubations were carried out in a controlled environment: humid atmosphere of 80 - 100% (actual range 49 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 – 37.2°C).

TEST FOR INTERFERENCES
- The test substance was checked for possible color interference and reduction of MTT before the study. No interference was found.

APPLICATION OF TEST ITEM
Before application of the test substance, the tissues were transfered to 6 wells plates with DMEM 0.9% and incubated for approximately 2 hours. The skin was moistened with 25 μL Milli-Q water to ensure close contact of the test item to the tissue and the solid test item was added into the 6-well plates on top of the skin tissues. For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MEASUREMENT OF CELL VIABILITY
- MTT concentrate (5 mg/mL) was diluted in a proportion 1:5 with supplemented DMEM: MTT-medium
- MTT-medium was added to the wells (300 μL)
- Incubation time: 3 hours at 37°C in air containing 5% CO2.
- Formazan was extracted with 2 mL isopropanol (MatTek corporation) overnight at room temperature.
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.


DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with
the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

- 51.4 to 52.7 mg of the solid test item was added into the 6-well plates on top of the skin tissues
- Positive control: (8N KOH): 50 μL
- Negative control: (Milli-Q water): 50μL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours incubation with MTT-medium

Number of replicates:

2 replicates exposed for 3 minutes
2 replicates exposed for 1 hour

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

115

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

OTHER EFFECTS
- Color Interference by the Test Item: no
- Reduction of MTT by the Test Item: no

ACCEPTANCE OF RESULTS
- The in vitro skin corrosion test is considered acceptable because the following criteria have been met:
a) The absolute mean OD570 of the two tissues of the negative control is within the laboratory historical control data range
b) The mean relative tissue viability following 1-hour exposure to the positive control is 10% (should be <15 %)
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates is <6% (should be ≤ to 30%)

Any other information on results incl. tables

Table 1 Mean Tissue Viability in the in vitro Skin Corrosion Test with SDH

	3-minute application viability (% of control)	1-hour application viability (% of control)
Negative control	100	100
SDH	115	92
Positive control	8.6	10

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin corrosion test was performed according to OECD guideline 431 and in accordance with GLP principles. The results show that SDH is non-corrosive to the skin, therefore, the substance does not need to be classified according to GSH and CLP criteria.

Executive summary:

An in vitro skin corrosion test using a human skin model (EpiDerm EPI200) was performed according to OECD/EC guidelines and in accordance with GLP principles. The test substance was applied directly to 0.6 cm²cultured skin (51.4 to 52.7 mg, in presence of 25μl Milli-Q water). After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. The positive control had a mean relative tissue viability of 10% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptancelimit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <6%, indicating that the test system functioned properly.

Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with SDH compared to the negative control tissues was 115 % and 92 %, respectively.

Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15 % after the 1-hour treatment, it can be concluded that the test substance is not corrosive in the in vitro skin corrosion test.