4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2016 to 19 May 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP

Test material

Test material information

Constituent 1

Reference substance name:

4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

EC Number:

276-602-4

EC Name:

4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Cas Number:

72363-26-9

Molecular formula:

C25H24N2O7S

IUPAC Name:

4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Details on test material:

Identification: FAT 40444/B
Lot: PCR92X140707(China)
Appearance: red powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sponsor Batch Number PCR92X140707 (China)
- Expiration date of the lot/batch:14 October 2019
- Purity: 95.4 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 °C in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Cell source:

other: No specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit from MatTek

Source strain:

other: Not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recognised in vitro test for corrosivity

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 23337
- Delivery date: 17 May 2016
- Date of initiation of testing: 17 May 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates: 2
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

- The test substance is considered to be non-corrosive to skin if [complete, e.g. 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 (volume or weight with unit): 25 mg, used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Sterile distilled water
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8.0N Potassium hydroxide

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

Duplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

107.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minute exposure

Value:

98.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.701 for the 3 Minute exposure period and 1.906 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.4% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

The relative mean ciabilities for each treatment group were:

Exposure period	Percentage viability
	Negative control	Positive control	Test item
3 minutes	100*	3.5	107.3
60 minutes	100*	3.4	98.7

 

*The mean viability of the negative control tissues is set to 100 %.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40444/B was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of FAT 40444/B using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item. The study was carried out according to OECD guideline 431 and EU method B.40. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item produced a sufficient amount of color that may interfere with the end point optical density readings. Therefore, additional tissues were incorporated into the testing for color correction purposes. These tissues were treated identically with the exception of not being placed onto MTT. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viabilities for each treatment group were as follows:

 

Exposure period	Percentage viability
	Negative control	Positive control	Test item
3 minutes	100*	3.5	107.3
60 minutes	100*	3.4	98.7

*The mean viability of the negative control tissues is set to 100 %.

The quality criteria required for acceptance of results in the test were satisfied. So based on the study results, FAT 40444/B was considered to be non-corrosive to the skin.