1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2021 to 05 June 2021

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2020

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 131 (09 June 2012): EpiSkinTM Skin Irritation Test 15 min – 42 hours

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

EC Number:

241-443-1

EC Name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Cas Number:

17418-59-6

Molecular formula:

C22H17NO5

IUPAC Name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Details on test material:

Physical state: Red powder
Storage condition: at room temperature

In vitro test system

Test system:

human skin model

Remarks:

EpiSkin™ Small Model (EpiSkin™SM)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: See below

Source strain:

other: Adult human derived

Details on animal used as source of 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.: 21-EKIN-022
Expiry date: 07 June 2021

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 ON THE 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Tissue batch number(s): 21-EKIN-022
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 03 June 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.9-23.7 °C)
- Temperature of post-treatment incubation (if applicable): 37±1 °C

NUMBER OF REPLICATE TISSUES: Three replicates were used for the test item and positive and negative controls.
Additionally, 2 replicates of colour controls (NSCliving), 2 replicates of non-specific colour control (NSCkilled), 3 killed test item treated tissues and 3 killed negative control treated tissues were used for the MTT evaluation.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL 1x PBS (phosphate buffered saline) solution

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): Place the living epidermis in a 12 well plate with 2 mL of distilled water (replacing the culture medium). Incubate at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere for 48 h +/- 1 hours. At the end of the incubation, discard the water.
- N. of replicates : 3 killed test item treated tissues and 3 killed negative control treated tissues
- Method of calculation used:
Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 5 (Test Item, Positive and negative control, Additional controls for MTT direct interacting chemicals, Additional controls for dyes and chemicals able to colour the tissue and Additional controls for non-specific colour in killed tissues)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritating to skin if the viability after 15 minutes exposure is less than 50% of the negative control

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: yes, for the non-specific OD evaluation (NSCliving), and also to detect and correct for test item interference with the viability measurement

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 10 μL (1x PBS)

POSITIVE CONTROL
- Amount(s) applied: 10 μL (SDS 5%)

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 h)

Number of replicates:

In this assay 3 replicates of test item, 3 replicates of negative control, 3 replicates of positive control, 2 replicates of colour controls (NSCliving), 2 replicates of non-specific colour control (NSCkilled), 3 killed test item treated tissues and 3 killed negative control treated tissues were used for the MTT evaluation.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 runs

Value:

75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrected relative viability

Run / experiment:

Mean of 3 rubs

Value:

71

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: Not significant
- Colour interference with MTT: Not significant

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was 1.310 and the % tissue viability was 100%.
- Acceptance criteria met for positive control: Yes. The mean OD value obtained for the positive control was 0.059 and this result corresponds to 4 % viability.
- Acceptance criteria met for variability between replicate measurements: Yes. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Any other information on results incl. tables

OD values and viability percentages of the positive and negative controls and test material

Substance	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Negative control: 1x PBS	1	1.408	-	108	-
	2	1.311	-	100	-
	3	1.210	-	92	-
	mean	1.310	-	100	-
	standard deviation (SD)			7.57	-
Positive Control: SDS (5 % aq.)	1	0.049	-	4	-
	2	0.048	-	4	-
	3	0.079	-	6	-
	mean	0.059	-	4	-
	standard deviation (SD)			1.32	-
Test Material	1	0.973	0.918	74	70
	2	1.158	1.102	88	84
	3	0.826	0.771	63	59
	mean	0.986	0.930	75	71
	standard deviation (SD)			12.69	12.69

 

INDICATOR FOR POTENTIAL FALSE VIABILITY

Possible direct MTT reduction with test material - As the test material has an intrinsic colour (red), the check-method for possible direct MTT reduction with test material was not completed for the following reason: if the test material has dark colour (red, green, blue ect.), the additional controls were used automatically. This step prevents the false viability which is the possible interaction with MTT, and it occurs if the test material cannot be removed totally from the surface of the tissues. In this situation the direct interaction with MTT is defined based on the OD results of additional controls. The non-specific MTT reduction (NSMTT) was determined to be 4 %. As the NSMTT was below 50 %, the true MTT metabolic conversion and the correction of viability percentages were undertaken.

Colouring potential of test material - The test material has an intrinsic colour (red). If the test material is not white, off white, almost white and/or colourless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test material. Two additional test material-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.046. The Non Specific Colour % (NSCliving %) was calculated as 3 % (below 5 %). Therefore, additional data calculation was not necessary. A false estimation of viability can be precluded.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

In this in vitro skin irritation test using the EPISKIN model, the test material did not show significantly reduced cell viability in comparison to the negative control (mean corrected relative viability value: 71 %). All obtained test material viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore, the test material was considered to be non-irritant to skin under the conditions of this study.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B. 46 in accordance with GLP using the EPISKIN Model.

Using this method, a small amount of the test material was applied to the surface of a three-dimensional human epidermis model (EpiSkin) and the optical density (OD) measured after an incubation period, from which the relative cell viability could be calculated. Additionally, positive and negative controls were performed, as well as controls for possible MTT-interacting items and chemicals able to colour the tissue.

Since the % relative tissue viability was calculated to be >50% for the test item, under the conditions of this study the test material was not irritating.