2-(4-chlorobenzoyl)benzoic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 14, 2016 to September 16, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chlorobenzoyl)benzoic acid

EC Number:

201-615-9

EC Name:

2-(4-chlorobenzoyl)benzoic acid

Cas Number:

85-56-3

Molecular formula:

C14H9ClO3

IUPAC Name:

2-(4-chlorobenzoyl)benzoic acid

Test material form:

solid

Details on test material:

Purity >99.1 %; Appearance: white powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKINTM Small Modell (SM)

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin
EPISKINTM (SM) (Manufacturer: SkinEthic, France), is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

As the test substance was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test substance
and epidermis and then 10 mg of the test substance was applied evenly to the epidermal surface.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 of the test substance, negative and positive control. Furthermore, as the test substances were coloured, two additional test substance-treated tissues were used for the non-specific OD
evaluation.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

107.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

- As no colour change (yellow colour) was observed after three hours of incubation of the test substance in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

- As the test substance was coloured, two additional test substance-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.013, Non Specific Colour % was calculated as 1.9 %. This value was

below 5 %, therefore additional data calculation was not necessary.

- The OD values for the test substance treated skin samples showed 107.1 % relative viability.

Applicant's summary and conclusion

Interpretation of results:

other: CLP

Remarks:

non-irritant according to CLP

Conclusions:

Under the study conditions, the test substance is not irritant to the skin in in vitro EPISKIN model.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance using the Reconstructed Human Epidermis (RhE) Test according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. The test substance, as well as controls, were tested for their ability to impair cell viability in the EPISKIN model after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The test substance was tested as supplied without vehicle. Three replicates of EPISKINTM (SM) disks were treated with the test substance. As the test substance was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface and then 10 mg were applied evenly to the epidermal surface and incubated for 15 minutes at room temperature. Exposure was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then further incubated for 42 hours at 37°C and 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C and 5% CO2. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test substance. For each treated tissue, the viability was expressed as a % relative to the negative control. Following exposure with the test substance, the mean cell viability was 107.1% compared to the negative control. This is above the irritancy threshold established for the test of 50%, therefore the test substance was considered as being non-irritant to skin. The experiment met all validity criteria, therefore the study was considered to be valid. Under the study conditions, the test substance was not irritant to the skin in thein vitroEPISKIN model (Kovács, 2017).