[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 11 2020 to November 13 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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

Reference substance name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Molecular formula:

Cannot be adequately assigned

IUPAC Name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Molecular formula: C50H52CuN10Na2O20S8 (main component)
Molecular weight: 1479.02 (main component)
Purity of test item: 93.1%
Impurity
Water: 6.5%
EDTA-4Na: 0.4%
Supplier: FUJIFILM Corporation
Lot number: M-002P
Storage conditions: Store in the dark at room temperature.

Specific details on test material used for the study:

Not specified

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes

Cell source:

other: Manufactured by EPISKIN Laboratories Lyon, France, is a three-dimensional human epidermis model.

Source strain:

other: Adult human-derived epidermal keratinocytes

Details on animal used as source of test system:

Not applicable.

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN Laboratories Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Supplier: EPISKIN Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 20-EKIN-046
Expiry date: 16 November 2020

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 mg of the test item
10 μL positive control
10 μL negative control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Dye-2019

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Criteria met
- Acceptance criteria met for positive control: Criteria met
- Acceptance criteria met for variability between replicate measurements: Criteria met
- Range of historical values if different from the ones specified in the test guideline:
- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction:
- Colour interference with MTT:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:
- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction:
- Colour interference with MTT:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline:

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Dye-2019 is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

EpiSkinTM SM test of Dye-2019 has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 26 June 2020.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with 1xPBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.
The test item has an intrinsic colour (blue), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).
The test item is a possible MTT-reducer, therefore additional controls (three test item treated killed tissues and three negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.
The test item is a possible MTT-reducer and has an intrinsic colour (blue). To avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. However, the NSCliving % was 2% (below 5 %), the NSCkilled was not determined and used during the calculation of true MTT metabolic conversion.
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
In this in vitro skin irritation test using the EPISKIN model, the test item Dye-2019 did not show significantly reduced cell viability in comparison to the negative control (mean corrected relative viability value: 90 %). All obtained test item viability results were far above 50 %, when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model (OECD 439), indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Dye-2019 is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).