2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU Method B.40-BIS

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

EC Number:

612-028-6

Cas Number:

607724-47-0

Molecular formula:

C26H22N5Na3O16S5

IUPAC Name:

2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

Test material form:

solid: particulate/powder

Details on test material:

1 MATERIALS AND METHODS
1.2 Test Item
Designation in Test Facility: 17031402G
Date of Receipt: 14. Mar. 2017
Condition at Receipt Room temperature, in proper conditions
1.2.1 Specification.
Name Blendazol Red Blendwell
Batch no. E 328
Appearance Dark Red Powder
Composition 2-Naphtalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, triso dium salt
Purity 95 % by HPLC
Homogeneity homogeneous
Expiry date 17. Feb. 2019
Storage Room Temperature: (20 ± 5°C); Keep away from humidity
CAS No. 607724-47-0
EINECS-No. 612-028-6
Chemical Class not stated
Stability H2O: 96h; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown
Solubility H2O: to be determined*; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown


* Will be determined in another GLP-study at LAUS GmbH and will be stated in the final report, this
This information is not provided by the sponsor but determined at LAUS GmbH.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: The test system is a commercially available EpiDermTM-Kit, procured by MatTek.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cul-tures inserts
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Designation of the kit: EPI-200-SCT
Day of delivery: 16. May 2017
Batch: 25812

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.4 mg
- Concentration (if solution):

VEHICLE MTT
- Amount(s) applied (volume or weight with unit):2 ml
- Concentration (if solution):1mg/L
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Demineralised water
- Concentration (if solution): 50µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL of KOH
- Concentration (if solution): 8M Solution in demineralised water

Duration of treatment / exposure:

3 min
1 h

Duration of post-treatment incubation (if applicable):

55 min

Number of replicates:

2 experiments

Test system

Type of coverage:

other: The test item was applied to each tissue and spread to match the tissue size.

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):25.4 mg +/-0.1mg
- Concentration (if solution):


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Demineralised water
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): KOH, CAS No. 1310-58-3, solution in demineralised water (8 M), batch no: 20150617.
- Concentration (if solution): 8M

Duration of treatment / exposure:

3MIN
1H

Details on study design:

TEST SITE
- Area of exposure: The test item was applied to each tissue and spread to match the tissue size.
- % coverage: 100%


REMOVAL OF TEST SUBSTANCE
- Washing (if done): Dulbecco’s Phosphate-Buffered Saline”; Solution was used for the rinsing of the tissues
- Time after start of exposure: 55 min

OBSERVATION TIME POINTS : “3 minutes” and “1 hour”


SCORING SYSTEM:
- Method of calculation:
The photometric absorbance of the negative controls was considered as 100%. For the mean of the 3 replicates of test item and positive control, tissue viability was calculated as % photometric absorbance compared to the negative control.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results

% tissue viability = (OD 1 replicate test item resp. positive control)*100%
OD mean of negative controls


1OD= Optical Density

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min/ 1 hour

Value:

>= 93 - <= 109.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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:- OTHER EFFECTS:
- Visible damage on test system: NO
- Direct-MTT reduction:
- Colour interference with MTT: NO

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: opti-cal density was 1.8 (3 minutes) resp. 1.9 (1 hour).
- Acceptance criteria met for positive control:
The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The viability was 8.4 %
- Acceptance criteria met for variability between replicate measurements: YES

Any other information on results incl. tables

For the test item and the positive control, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative controls:

Table8.2‑a      % Tissue Viability

Test Item	Positive Control	Incubation
109.7%	23.2%	3 min
93.0%	8.4%	1 h

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

non-corrosive to skin.

Conclusions:

The relative absorbance values of the test item were increased to 109.7% after 3 minutes treatment. This value is above the threshold for corrosivity (50%). After 1 hour treatment, the relative absorbance values of the test item were reduced to 93.0%, lying above the threshold for corrosivity (15%). Therefore, the test item is considered as non-corrosive to skin.

Executive summary:

The Determination of Skin Corrosion Potential of Blendazol Red Blendwell in the Reconstructed Human Epidermis (RhE)

Test Method was determined following OECD Guideline 431and EU-Method B.40-BIS

One valid experiment was performed.

Two tissues of the human skin model EpiDerm^TMwere treated with the test itemBlendazol Red Blendwell for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.8 (3 minutes experiment) and 1.9 (1 hour experiment). The positive control showed clear corrosive effects for both treatment intervals. The relative absorbance value was reduced to 8.4 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the relative absorbance values were increased to 109.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, relative absorbance values were reduced to 93.0 %. This value, too, is above the threshold for corrosion potential (15%). In the guideline, values greater or equal to the threshold are considered as “non-corrosive to skin”.

 

Therefore, Blendazol Red Blendwell is considered as

non-corrosive to skin in the Reconstructed Human Epidermis (RhE) Test Method