Reaction products of Hydrogen amino-4-hydroxynaphthalene-2-sulfonate, Potassium substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate and Sodium substituted-({4-[(2-chloroethyl)sulfonyl]butanoyl}amino)benzenesulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Test item : FAT 40875/A TE
Physical appearance : Dark red powder
Purity as per CoA : 86.4% all organic components
Batch No. : BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured date : 20 December 2017
Expiry date : 11 December 2022
pH : 4.3 (aq. soln. 2 %w/w at room-temperature)
Recommended storage condition: Refrigeration (+2 to +8 °C) and protection from light

Specific details on test material used for the study:

Test Item: 40875/A TE
Physical Appearance: Dark red powder
Purity as per Certificate of Analysis (Content): 86.4% all organic components
Batch No.: BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured Date: 20 December 2017
Expiry Date: 11 December, 2022
Recommended Storage Condition : Refrigeration (+2 to +8 °C), Protect from light
pH: 4.3 (aq. soln.(2% (w/w) at room-temperature)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal human keratinocytes

Justification for test system used:

The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals. One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 20 - 28 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 42h
- Spectrophotometer: TECAN Infinite® M200 Microplate Reader
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: Triplicate

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if [complete, e.g. 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 [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:

10 mg/tissue of test substance was applied topically to the EpiSkin three dimensional RhE model.

Negative control : 10 microliter of Phosphated Buffered Saline (PBS).
Positive control : 10 microliter of 5% Sodium Dodecyl Sulfate (SDS).

Duration of treatment / exposure:

Incubation for 15 minutes at room temperature (20 - 28 °C)

Duration of post-treatment incubation (if applicable):

Tissues were incubated with MTT solution for 4 hours and then extracted with acidified isopropanol to quantify spectrophotometrically at wavelength of 570 nm.

Number of replicates:

Three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

True tissue viability

Value:

131.23

Vehicle controls validity:

valid

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: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 0.753% mean cell viability.
- Acceptance criteria met for positive control: 6.89% mean cell viability.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation was within the range.

Any other information on results incl. tables

Individual tissue viability of epidermis units (relative)

	% Individual Viability
	R1	R2	R3	Mean	SD
Positive control	6.63	7.40	6.64	6.89	0.44
Test item	88.07	88.30	91.89	89.42	2.14
% NSC living tissue	4.13	4.30	-	4.22	0.12
% NSMTT killed tissue	4.86	6.61	-	5.74	0.24
% NSC killed tissue color	51.21	52.32	-	51.77	0.78

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for the test item, FAT 40875/A TE was 131.23 % and hence, the test item is predicted to be non-irritant.

Executive summary:

The test item, FAT 40875/A TE was tested for its skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. This test was conducted in accordance with OECD test guideline 439 in a GLP certified laboratory.

 

10 mg/tissue of test item, 10µL of negative control (PBS) & 10µL of positive control (5% of SDS) respectively was exposed for 15 minutes.

In the preliminary tests, FAT 40875 was identified as producing direct MTT reduction and color interference and thus adapted controls evaluation was performed.

After approximately 42-hour post incubation period, irritation potential was evaluated by assessing the cytotoxic (irritancy) effect. The cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured on formazan production from MTT and the end of treatment.

Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 mins of treatment with the test item is compared with negative control tissues.

The absolute mean Optical density (OD) at 570nm of negative control, positive control and test item was 0.7530, 0.0519 and 0.6733 respectively. The positive control had a mean cell viability of 6.89% after 15 mins exposure, indicating the test system functioned properly.

The true tissue viability of the test item was found to be 131.23%.

The study indicated that the test item FAT 40875/A TE is predicted to be non-irritating in this In-vitro skin irritation test using Reconstructed Human Epidermis under the condition of testing employed.