2-(Hydroxyacetyl)-N-methylhydrazine-1-carboxamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th June 2017 to 14th July 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

MatTek EpiDerm™ MTT Viability Assay

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-N-[(methylcarbamoyl)amino]acetamide

EC Number:

815-593-9

Cas Number:

1613307-26-8

Molecular formula:

C4H10N3O3

IUPAC Name:

2-hydroxy-N-[(methylcarbamoyl)amino]acetamide

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

other: MTT solution (1 mg/ml methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium [DMEM]).

Details on test system:

EpiDerm™ Tissue Samples
EpiDerm™ tissues, Lot 26490 Kit D, were received from MatTek on 11 Jul 2017 and refrigerated at 2-8ºC. Before use, the tissues were incubated (37oC ± 1oC, 5% ± 1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.
Test Article Reduction of MTT
100 mg of the test article were mixed with 1 ml of MTT solution (1 mg/ml methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium [DMEM]). A negative control, undosed issues, was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than its actual irritation potential. The test article did not reduce MTT and the assay continued as per the protocol.
Dosing
At the request of the Sponsor, the test article was dosed neat. 100 mg of the test article were applied to the top of each EpiDerm™ tissue. The test article remained in contact with the EpiDerm™ tissue for 1, 4 and 24 hours. A negative control (undosed tissues) was tested in the same manner at 4 hours. A positive control (100 μl of 1% Triton® X-100) was tested at 4 and 9 hours. Each treatment with test article or control was conducted in duplicate.
Tissue Viability (MTT Reduction)
At the end of the selected exposure periods, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 μl of MTT solution. The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then transferred to another 24-well plate containing 2.0 ml of extractant solution (isopropanol) per well. The tissues were then incubated overnight in the dark at room temperature. An aliquot of the extracted MTT formazan was measured at 540 nm using a plate reader, subtracting the absorbance at a reference wavelength of 690 nm.

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

100 μl of 1% Triton® X-100

Duration of treatment / exposure:

The solutions were incubated at room temperature in the dark for 60 minutes

Duration of post-treatment incubation (if applicable):

At the end of the selected exposure periods, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well plate containing 300 μl of MTT solution. The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then transferred to another 24-well plate containing 2.0 ml of extractant solution (isopropanol) per well. The tissues were then incubated overnight in the dark at room temperature.

Number of replicates:

Each treatment with test article or control was conducted in duplicate.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 24

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

MatTek EpiDerm™ tissue samples were treated in duplicate with the test article, and positive control for various exposure times listed below. A negative control (undosed tissues) was tested at 4 hours only. Following treatment, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm using a reference wavelength of 690 nm. The viability was then expressed as a percent of negative control values. The mean percent viability for each time point was used to calculate an ET₅₀, which represents the time at which the EpiDerm™ tissue viability was reduced 50% compared to control tissues. The ET₅₀ scores were converted to an irritancy classification. 

The ET50 (hr) of L -005313063-000D009 exposed for 1, 4 and 24 hours was >24.0, which gave an irritancy classification of Non-Irritating