Methyl 4-[2-[4-(5-methyl-2-benzoxazolyl)phenyl]vinyl]benzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April from 25th to 27th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fluorescent Brightener 371

IUPAC Name:

Fluorescent Brightener 371

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

- System: EpiSkinTM Small Model (EpiSkinTMSM), a three-dimensional human epidermis model.
- Supplier: SKINETHIC Laboratories, France
- Source: adult human-derived epidermal keratinocytes.
- Seeding and culturing: 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.
- Quality Control: EpiSkinTMSM 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 acceptability range established by the RhE model developer/supplier is 1.5 mg/l ≤ IC50 ≤ 3 mg/l, in accordance with those indicated into the OECD guideline.
- Storage: the EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8 °C.

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-INCUBATION (day [-1]-0)
The maintenance medium was pre-warmed to 37 °C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 ml per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (18-24h) at 37 ± 1 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere.

PRE-APPLICATION (day 0): the epidermal surface was first moistened with 5 µl deionised water.

RINSING (day 0): with approximately 25 ml PBS 1x. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT TEST AFTER INCUBATION (day 2): after post-incubation period the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 ml of 0.3 mg/ml MTT per well) and then incubated for 3 hours (± 5 min) at 37 ± 1 °C in an incubator with 5 ± 1 % CO2 protected from light, ≥ 95 % humidified atmosphere.

FORMAZAN EXTRACTION
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µl acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 µl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm (± 10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µl).

CONTROLS
- Ngative control: a volume of 10 µl 1x PBS was applied on the skin surface by using a suitable pipette.
- Positive: a volume of 10 µl SDS 5 % aq. was applied on the skin surface by using a suitable pipette.

CHECK-METHOD FOR DIRECT MTT REDUCTION
- Solution: approximately 10 mg test item was added to 2 ml MTT solution (diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/ml) and mixed.
- Incubation: the mixture was incubated for three hours at 37 ± 1 °C protected from light.

CHECK-METHOD FOR COLOURING POTENTIAL
- Solution: 10 mg test item was added to 90 μl of water and mixed.
- Treatment and observation: the mixture was shaken for 15 minutes at room temperature and then colour checked (unaided eye assessment).

ADDITIONAL CONTROLS
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.

INTERPRETATION OF RESULTS
- Mean tissue viability % is ≤ 50 %: substance skin irritating.
- Mean tissue viability % is > 50 %: substance non skin irritating.

ACCEPTANCE CRITERIA
- The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40 % and the standard deviation value (SD) of the % viability should be ≤ 18.
- For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

The plates were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Duration of post-treatment incubation (if applicable):

The plates were incubated for 42 hours (± 1h) at 37 ± 1 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere.

Number of replicates:

3 replicates per test item, 3 replicates negative controls, 3 replicates positive controls and 2 replicates colour controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates

Value:

85

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 85 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

CHECK-METHOD FOR DIRECT MTT REDUCTION
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

CHECK-METHOD FOR COLOURING POTENTIAL
The test item showed no ability to become coloured in contact with water. However, the test item has an intrinsic colour (yellowish), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.047. The Non Specific Colour % (NSC %) was calculated as 7.1 % (above 5 %). Therefore additional data calculation was necessary.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.662. The mean OD value obtained for the positive control was 0.159 and this result corresponds to 24 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Any other information on results incl. tables

OD values and viability percentages of the test item (including corrected values)

Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
1	0.543	0.496	82	75
2	0.585	0.538	88	81
3	0.697	0.650	105	98
mean	0.608	0.562	92	85
standard deviation (SD)			12.01	12.01

TOD_TT: true MTT metabolic conversion

OD values and NSC % of additional control

Optical Density (OD)		Non Specific Colour % (NSC %)
1	0.047	7.1
2	0.047
mean	0.047

OD values and viability percentages of the controls

Substance	Optical Density (OD)		Viability (%)
Negative Control	1	0.604	91
	2	0.761	115
	3	0.621	94
	mean	0.662	100
	standard deviation (SD)		13.04
Positive Control	1	0.143	22
	2	0.236	36
	3	0.098	15
	mean	0.159	24
	standard deviation (SD)		10.65

Applicant's summary and conclusion

Interpretation of results:

other: not classified. according to the CLP Regulation (EC) 1272/2008

Conclusions:

Non skin irritating

Executive summary:

EpiSkinTM SM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1h) in an incubator with 5±1 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item showed no ability to become coloured in contact with water. However, the test item has an intrinsic colour (yellowish), two additional test item-treated tissues were used for the non-specific OD evaluation.

Under the tested conditions, the test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 85 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin.

Conclusion

Non skin irritating