Monalazone disodium

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Monalazone disodium

EC Number:

262-810-2

EC Name:

Monalazone disodium

Cas Number:

61477-95-0

Molecular formula:

C7H4ClNO4S.2Na

IUPAC Name:

disodium 4-[(chloroazanidyl)sulfonyl]benzoate

Specific details on test material used for the study:

20% Monalazone Disodium
5% Monalazone Disodium in water.
Stored at room temperature in the absence of light. The dilutions were prepared immediately prior to exposures.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

The 3-Dimensional human epithelial skin model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes cultured on a permeable synthetic membrane at the air-liquid interface in a chemically defined medium. The cells form a multilayered, highly differentiated model of the human epidermis that consists of organized basal, spinous, granular, and cornified layers and closely resembling native epidermis. Each lot of tissues is Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).

Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for one hour. The tissues were then transferred to 6-well plates containing 0.9 mL of fresh Maintenance medium and they were incubated overnight. 30 µL of the negative control, DPBS, and positive control, 5% SDS, were added to the apical surface of tissues. 30 µL of each test material was added to the apical surface of tissues. All tissues were placed into the 37°C incubator with 5% CO2. The exposure time was 1 hour, with 35 minutes exposure in the incubator and 25 minutes at room temperature. After the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 hours. After 24 hours, the basal media was replaced with 0.9 mL of fresh Maintenance medium (6-well plate) and incubated for another 18 hours prior to performing the MTT assay.

Tissues were then evaluated for cell viability using an MTT Assay where yellow MTT is reduced to purple formazan primarily by enzymes (reductases) located in the mitochondria of living cells. According to established Cyprotex procedure, a stock solution of MTT (Sigma, M-5655) was prepared in Maintenance medium (provided with tissues) just prior to use and warmed to 37°C in a water bath. Tissues inserts were transferred to 24-well plates containing 300 µL MTT medium (1 mg/mL). After 3 hour MTT incubation, the formazan salt was extracted with 2 mL isopropanol per tissue and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Viable cells had the greatest amount of MTT reduction and hence the highest absorbance values. Relative cell viability was calculated for each tissue as % of the mean of the negative control tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 ul

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Three

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

20% Monalazone disodium

Value:

11.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

5% Monalazone Disodium

Value:

42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

	Mean	SD	SEM	%CV	p-value	Significant?
DPBS	100.0	3.6	2.1	3.6
5% SDS	5.0	0.7	0.4	13.1	<0.001	YES
20% monalazone disodium	11.9	5.2	3.0	43.7	<0.001	YES
Dilute (5% monalazone disodium) in Water	42.0	8.3	4.8	19.8	0.001	YES

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test articles (20% monalazone disodium and 5% monalazone Disodium) were determined to be irritants to human skin as the viability of the exposed cells were <50% of control.

Executive summary:

The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article under three conditions by determining the viability of the tissues following exposure via MTT. The objective of this study was to assess the dermal irritation potential of the Sponsor’s submitted test article under three conditions. Pre-testing showed the test articles/conditions were not colored though one test article, 20% Monalazone Disodium did auto-reduce MTT. A small data correction was necessary as the auto-reduction was observed during the study. Tissues were exposed to test articles and controls for one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.

The assay passed all the quality controls. The negative control tissue OD was between 1.651 and 1.942, the positive control was within the confidence limits and was deemed an irritant, and the test article SDs were all ≤18. The MTT data show the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 5.0% of control. The mean viability of tissues after exposure to the test articles, 20% monalazone disodium and Dilute 5% monalazone disodium in water were <50% of control. Therefore, the test articles were determined to be irritants to human skin according to the OECD test guideline followed for this study.