Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

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 4 March 2014 to 10 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Results from water killed tissues were not used; these deviation was considered not to affect the integrity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Vivimed, lot no. 20140101
- Expiration date of the lot/batch: January 2016
- Purity test date: 16th January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature in the dark
- Stability under test conditions: not specified, test item used pure
- Solubility and stability of the test substance in the solvent/vehicle: not specified, test item used pure

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied

In vitro test system

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

other: Reconstrcted Human Epidermis EPISKINtm

Cell source:

other: Reconstrcted Human Epidermis EPISKINtm

Source strain:

not specified

Justification for test system used:

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM
- Tissue batch number(s): 14-EKIN-007
- Production date: not specified
- Shipping date: not specified
- Delivery date: 4 March 2014
- Date of initiation of testing: 4 March 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:37 deg Celsius
- Temperature of post-treatment incubation (if applicable): 37 deg Celsius

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one washing step after the 15 minutes exposure period with DPBS with Ca++ and Mg++
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: Not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:0.3mg/mL
- Incubation time:3 hours at 37 deg Celsius
- Spectrophotometer: Anthos 2001
- Wavelength:562nm
- Filter:no reference filter used
- Filter bandwidth: no filter used
- Linear OD range of spectrophotometer:not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No historical data provided

NUMBER OF REPLICATE TISSUES: triplicates per conditions

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Water killed tissues used
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared by placing untreated EpiSkinTM tissues in a 12 well plate containing 2.0 mL distilled water in each well. The tissues were incubated at 37 deg Celsius 5% CO2 in air for 48±1 hours. At the end of the incubation time the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 deg Celsius) for up to 6 months. Before used each tissue was thawed by placing in 2.0 mL of maintenance medium due to residual reducing enzymes within the killed tissues.
- N. of replicates : triplicate
- Method of calculation used: Relative mean viability (%) = (mean OD562 of test item / mean OD 562 of negative control ) x100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 sequencies : pre incubation and main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post exposure period is less than 50%,
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post exposure period is greater than
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)
- Concentration (if solution): used pure

VEHICLE
no vehicle, 5µL of distilled water was applied on topically applied to improve the contact between skin and the test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): used pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Test : Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Test : Positive control

Value:

7.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Test : Test Item

Value:

96.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: No direct reduction
- Colour interference with MTT: No interference

DEMONSTRATION OF TECHNICAL PROFICIENCY: SDS used as positive control

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
- Range of historical values if different from the ones specified in the test guideline: Not specified

Any other information on results incl. tables

Table1 :Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD562 of tissues	Mean of OD562	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability(%)
Negative Control	0.914	0.915	0.030	99.9	100*	3.3
	0.886			96.8
	0.945			103.3
Positive Control	0.095	0.071	0.021	10.4	7.7	2.3
	0.059			6.4
	0.046			6.4
Test Item	0.829	0.879	0.044	90.6	96.1	4.8
	0.897			98.0
	0.912			99.7

*set at 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, the test item Acid Violet 43 did not induce irritation to skin sample EpiSkinTM after 15 minutes exposure period and 42 hours post exposure period (96.1% of viability). Hence, the substance was considered as Not Irritating to skin according CLP regulation.

Executive summary:

The purpose of this GLP compliant test was to evaluate the skin irritation potential of the test item using the EpiSkinTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours according to the OECD guideline 439 method.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. Additional non-viable tissues were incorporated into the testing for correction purposes to MTT interferences (Water-killed tissues). At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

The relative mean viability of the test item treated tissues was 96.1% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Under the experimental conditions of the study, incubation of the registered substance Acid Violet 43 led to a relative mean viability at 63.3% compared to negative control. Hence, the registered substance was classified as Non-irritant (EU CLP Not classified for Irritation).