Ethyl 9-oxo-9H-thioxanthene-2-carboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 - 16 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 9-oxo-9H-thioxanthene-2-carboxylate

EC Number:

280-960-7

EC Name:

Ethyl 9-oxo-9H-thioxanthene-2-carboxylate

Cas Number:

83817-60-1

Molecular formula:

C16H12O3S

IUPAC Name:

ethyl 9-oxo-9H-thioxanthene-2-carboxylate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Rahn AG, Zürich, Switzerland

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dry conditions. Protect from heat and direct sunlight.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing. It is used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and non-skin irritating test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number(s): 16-EKIN-037
- Date of initiation of testing: 14 Sep 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume of washing steps: rinsed thoroughly with 25 mL PBS 1x solution
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 21.8 ± 0.6
- Barrier function: 1.9 mg/mL
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Method of calculation used:
Blank: The mean of the 6 blank OD values is calculated
Negative control:
- Individual negative control OD values are corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNC raw – ODblank mean
- The corrected mean OD of the 3 negative control values is calculated: this corresponds to 100% viability
Positive control:
- Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPCraw – ODblank mean
- The corrected mean OD of the 3 positive control values is calculated
- The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
- The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item:
- Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
- The corrected mean OD of the 3 test item values is calculated
- The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
- The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

As the test item has an intrinsic colour (pale yellow), further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.

Additional controls for dyes and chemicals able to colour the tissue:
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.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the mean relative viability after 15 minutes exposure and 42 h post incubation is less or equal to 50% of the negative control.
- The test substance is considered to be non-corrosive or non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more than 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 10 μL

POSITIVE CONTROL
- Amount(s) applied:10 μL
- Concentration: 5%

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of negative control (1x PBS)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of positive control (5% SDS)

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of test item (10 mg on 0.38 cm²)

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no
Colouring potential of test item: As the test item has an intrinsic colour (pale yellow), 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 0.032. The Non Specific Colour % (NSC %) was calculated as 3.6 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
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.

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: MTT assay after 3 hours (± 5 min) exposure

Substance	Tissue No.	OD570	OD570 (mean)	Relative viability [%]	Relative viability (mean) [%]	Standard Deviation of relative viability [%]
Negative Control	1	1.018	0.869	117	100	15.22
	2	0.823		95
	3	0.765		88
Positive Control	1	0.167	0.136	19	16	6.34
	2	0.168		19
	3	0.072		8
Test Item	1	0.718	0.835	83	96	12.30
	2	0.927		107
	3	0.862		99

Applicant's summary and conclusion

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified