Carboxylic acids, C5-9

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 July 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, C5-9

EC Number:

271-676-4

EC Name:

Carboxylic acids, C5-9

Cas Number:

68603-84-9

Molecular formula:

UVCB

IUPAC Name:

Carboxylic acids, C5-9

Test material form:

liquid

Details on test material:

Standard production of Matrica

In vitro test system

Test system:

human skin model

Cell source:

other: commercial reconstructed human epidermis (RhE) model, EPISKIN™. from human derived epidermis keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

MTT method: The test system EPISKIN™is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The principle of the RhE test method is based on the premise that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS N. 298-93-1] into a blue formazan salt that is quantitatively measured after extraction from tissues. Corrosive chemicals are identified by their ability to decrease cell viability below defined threshold levels.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL per well

Duration of treatment / exposure:

Exposure times of 3, 60±5 and 240±5 minutes were allowed in a ventilated cabinet at room temperature

Duration of post-treatment incubation (if applicable):

Plates were incubated for 3 hours ± 5 minutes at 37°C, 5% CO2 and saturated humidity

Number of replicates:

2

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 3 minutes of exposition

Run / experiment:

Main test

Value:

ca. 15

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

TABLE 1 - Preliminary Test

Direct MTT reduction test (Step1) 

Test item (mg)	MTT ready to use solution(mL)	Container	Incubation condition	Colour observation
50µL	2.0	well	3h at 37°C, 100% nominal humidity, 5% CO2	Opaque yellow solution with oily yellow drops and crystalline precipitate (no interaction)

 

 Colouring potential test (Step 2) 

Test item (mg)	Water (µL)	Container	Incubation condition	Colour Observation
10 µL	90	Eppendorf tube	15’, ambient condition, in agitation	Colourless solution (no interaction)

TABLE 2 - Main Assay - TREATMENT TIME: 3 minutes

			Test Item
							ODTI		Viability(%)
			TI1A-1 TI1A-2	0.146 0.154	0.1083 0.1163		0.1123		14.5
			TI1B-1 TI1B-2	0.153 0.162	0.1153 0.1243		0.1198		15.4

 

 

Mean	0.116	Mean	0.116		15
SD	0.007
CV(%)	5.6			∆(%)	6.5

Applicant's summary and conclusion

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

The test item induced cell death at all treatment times. Each mean cell viability, after the concurrent blank subtraction, was as follows:

Treatment time (minutes) Mean cell viability (%)
 3  15
 60 1
  240  2
Intra-replicate variability was acceptable with a difference of viability between the two replicates lower than 30%, for all treatment times.
Based on the results obtained, the test item MATRILOX IL001M is identified as corrosive to the skin (UN GHS Sub-category 1A).

Executive summary:

The potential of the test item MATRILOX IL001M to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. A preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. No interaction was recorded between the test item and MTT in test conditions similar to those of theMain Assay. In a second step, the test item was assayed for the ability of colouring water per se. No colouring potential was noted. Based on these results, no additional control were included in the main experiment. In theMain Assay, for each treatment time, the test item (physical state: liquid) was applied as supplied in two replicates, at the treatment level of 50 μL/epidermis unit, each measuring 0.38cm2 (treatment level: 131.6 μL/cm2). Positive and negative controls (Glacial acetic acid and Physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 μL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

In the Main Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability. The positive control caused the expected cell death (1% of cell viability, when compared to the negative control). Based on the stated criteria, the assay was regarded as valid.