Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion (Category 1, 1A, 1B or 1C)

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT) obtianed from MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic.
- Tissue batch number(s): 28644

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing with sterile PBS (filling and emptying of insert 20 times in a constant soft stream of PBS)
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Wavelength: 750 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissue were incubated with medium instead of MTT solution during the MTT incubation step.
- Method of calculation used:
Calculations for Data Correction Procedure for Colored Test Item:
OD = OD colored tissue (MTT assay) – OD colored tissue (no MTT assay)
In 3 minutes application, as the extract from tissues treated by colored substance (test item detected in step 1) has an OD <5 % of the negative control treated tissue, correction of the OD was not considered.
Correction of results were considered for 1 hour application as the OD of extract from tissues treated by colored substance was >5 % of the negative control treated tissue.

Check for direct MTT reducing substances:
To check whether test item directly reduces MTT or not, 25 mg of the test item was added to 1 mL of the MTT medium in a 6-well plate and incubated in the incubator (37±1 °C, 5±1 % CO2) for 60 minutes. Untreated MTT medium was used as control. Post incubation, MTT solution containing test item turned to dark blue color, hence considered as reducer of MTT. As test item reduced MTT, a functional check using freeze-killed tissue controls (killed controls = KC) was performed in one definitive assay to evaluate whether the test item is not binding to the tissue and leading to a false MTT reduction signal. All assay procedures are performed as for the viable tissue.

Calculations for Data Correction Procedure for MTT reducers:
True viability = Viability of treated tissue – Interference from test chemical
= OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue
kt = killed tissues
tkt = treated killed tissue
ukt = untreated killed tissue (NC treated tissue)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure (mean value of tissue 1 and 2)

Value:

108.87

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure (mean value of tissue 1 and 2)

Value:

13.44

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes. The test item is considered as MTT reducer as it formed dark black colour solution with MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The technical proficiency of the test method was established by using proficieney chemicals under Bioneeds Study No.: BIO-GT 1000. according to OECD test Guideline No. 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Summary of optical density (OD) and viability

3 Minutes Exposure

Treatment		OD	Viability (%)	CV (%)	Classification
Negative Control (Sterile water)	Mean	1.105	100.0	8.68	NC
	±SD	0.096	12.2
	n	2	2	2
Positive Control (Glacial acetic acid)	Mean	0.083	7.5	11.55	C (Category 1A)
	±SD	0.010	1.2
	n	2	2	2
Test Item	Mean	1.203	108.87	-	NC
	±SD	-	-
	n	2	2

 

OD (killed tissue)	=	Mean OD of treated killed tissue	-	Mean OD of untreated killed tissue
0.161	=	0.205	-	0.045
True Viability	=	OD of treated viable tissue	-	OD of killed tissue
1.042	=	1.203	-	0.161

 

1 Hour Exposure

Treatment		OD	Viability (%)	CV (%)	Classification
Negative Control (Sterile water)	Mean	1.838	100.00	0.40	NC
	±SD	0.007	0.6
	n	2	2	2
Positive Control (Glacial acetic acid)	Mean	0.065	3.6	1.08	C (Category 1A)
	±SD	0.001	0.1
	n	2	2	2
Test Item	Mean	0.197	7.40	-	C
	±SD	-	-
	n	2	2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation; CV = Coefficient of variation

 

 

Data Correction for Colored substance	=	OD of Colored tissue (with MTT)	-	OD of Colored tissue (Without MTT)
1.070	=	1.197	-	0.127
OD (killed tissue)	=	Mean OD of treated killed tissue	-	Mean OD of untreated killed tissue
0.822	=	0.890	-	0.067
True Viability	=	OD of treated viable tissue (MTT Assay)	-	OD of killed tissue
0.147	=	1.203	-	0.161

True viability (%) = 13.44%

Acceptance Criteria:

1. The assay met the acceptance criterion as the mean OD750 of the negative control tissues are 1.105 (after 3 minutes exposure) and 1.838 (after 1 hour exposure), which are in the range of ≥0.8 and ≤2.8.
2. The assay met the acceptance criterion as the mean viability of positive control tissues is 7.5 % (after 3 minutes exposure) and 3.6 % (after 1 hour exposure) which were ≤15 % of the negative control tissues and the SD of the three tissues replicates is 0.0 to 12.2.
3. The assay met the acceptance criterion as the OD of the extraction solvent was ≤0.1.
4. Two tissue replicates per exposure time was in range between 0.40 and 11.55 % viability the coefficient of variation (CV).

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Based on the results obtained under the conditions of this study, the test item is considered as corrosive to reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.

Executive summary:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated. The test item developed dark blue colour when dissolved in distilled water and isopropanol. The test item is considered as MTT reducer as it formed dark black colour solution with MTT. Hence, additional live and freeze killed tissue controls were maintained with and without MTT in duplicates. All assay procedures were performed as for the viable tissue but controls without MTT tissues were incubated with medium instead of MTT solution during the MTT incubation step.

Exposure to the test item was performed for 1 hour and 3 minutes separately. Tissues were treated with 25 mg test item or 50 µL of positive control (glacial acetic acid). At the end of treatment time, tissue inserts were rinsed with sterile PBS to remove any residual test item. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viabilily of tissues was calculated.

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±12.2, 7.5±1.2 and 108.87, respectively. Correction procedure for colorant control was not considered as the mean OD of extract from tissues treated by colored test item was less than 5 % of negative control. As the percentage viability of test item was greater than 50 % of the negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 15 % of negative control and clearly represents the irritation potential of positive control.

For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±0.6, 3.6±0.1 and 13.44. The percentage viability of the test item was less than 50 % of negative control. Therefore, the test item is considered as corrosive. The percentage viability of positive control (PC) is less than 15 % of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the conditions of this study, the test item is considered as corrosive to Reconstructed Human Epidermis (RhE) in accordance with UN GHS category 1, as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and less than 15 % after 1 hour exposure of the negative control.