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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28/05/2021-04/06/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted: 18th June, 2019; Corrected: 26 June 2020
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
31st July 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-chlorobenzoic acid
EC Number:
200-805-9
EC Name:
4-chlorobenzoic acid
Cas Number:
74-11-3
Molecular formula:
C7H5ClO2
IUPAC Name:
4-chlorobenzoic acid
Test material form:
solid: particulate/powder
Details on test material:
white to yellowish

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
approved method for non-animal testing under REACH
Details on test system:
Complementary Experiments
Direct MTT reduction
Functional check for this possibility was performed as follows: the test item was added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, humidified) for 60 minutes. At the end of the exposure time, the presence and intensity of the staining was observed.

Colour interference
Preliminary experiment was performed as follows: 25 mg of the test item were added to 2 ml isopropyl alcohol and placed on a shaker for 135 minutes at room temperature. Two 200 μL aliquots of isopropyl alcohol solutions and of pure isopropyl alcohol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength.
Another 25 mg of the test item were added to 0.3 mL of distilled water and kept at culture conditions for 60 minutes. Change of colour was evaluated by naked eye.

MTT test
a) Application:
25 mg of the test item was dosed directly on the tissue previously moistened with 25 μL of DPBS. The item was spread over the entire tissue surface. A single experiment, composed of three replicate tissues, was run.

b) Tissues preparation and treatment:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After 60 minutes of pre-incubation, medium was replaced and pre-incubation continued for other 19 hours and 9 minutes.
After pre-incubations, tissues were topically exposed to the test item for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.
After 24 hours of post-incubation period, the medium was replaced by a fresh one. Tissues were incubated for another 18 hours and 38 minutes.

c) MTT assay:
Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg·mL-1). After 180 minutes of MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for 164 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. Detailed procedure is described in internal SOP M/48/6 (VUOS-CETA, 2020).

d) OD570 measuring:
OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.
Control samples:
yes, concurrent negative control
yes, concurrent positive control

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
average
Value:
93.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
96.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
89.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
94.8
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Direct MTT Reduction - Functional Check in Tubes

After incubation of 25 mg of the test item with 1 mL MTT medium (red)for about 1 hour at culture conditions, MTT medium did not changed its colour towards yellow.

The test item did not reduce MTT directly.

Colour Interference

In the preliminary test, no change of colour was observed not in isopropyl alcohol neither in water. Average OD570of the test item dissolved in isopropyl alcohol was 0.0003, what is <0.08.After incubation of the test item with water, colouring also did not change. Colour of the test item did not interfere with the endpoint.

MTT Test

At incubation of the test item on the tissue surface, the test item was not wet in all its volume. The test item is presumed to be wetted on the tissue surface. No problems occurred at washing the test item off tissues and no changes of tissues were observed during the experiment.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The effect of the test item was negative in the EpiDermTM model.
According to the classification criteria, the test item is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant to skin.
Executive summary:

The test item, 4-chlorobenzoic acid, was assayed forin vitro skin irritation in the human epidermal model EpiDermTM. The main methodical documents were OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2019 and 2020), and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In the main MTT experiment, after pre-incubation of tissues, 25 mg of the test item was placed directly on the moistened tissue and spread over the entire surface for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used for the test item and for positive and negative controls.

After removal of the test item, tissues were post-incubated in culture medium for approximately 42 hours. After three hours incubation with MTT, samples were extracted with isopropyl alcohol for 2 hours and 32 minutes. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

No changes in appearance of tissues and its colour were observed during the MTT experiment.

In the complementaryexperiments, direct MTT reduction in test tubes was not found. Even the colour of the test item did not interfere with the evaluation of the test. Therefore, the results of the MTT test did not require correction.

Under the above-described experimental design, theaverage viability of test item-treated tissues was 93.6 ± 3.2%.

The positive control, SDS, gave the appropriate response.

 

The test item was negative in theEpiDermTM model (i.e., not irritating).

According to the classification criteria, the test item, 4-chlorobenzoic acid, is considered to have no category in regard to skin irritation.