1,2-dihydroxyanthraquinone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.11.2016 - 25.11.2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06-Jul-2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dihydroxyanthraquinone

EC Number:

200-782-5

EC Name:

1,2-dihydroxyanthraquinone

Cas Number:

72-48-0

Molecular formula:

C14H8O4

IUPAC Name:

1,2-dihydroxy-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: solid yellow powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

other: DPBS

Remarks:

To improve the contact between the test item and the epidermis

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia)
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.63 cm2
- Pre-incubation: 60 minutes (37 ± 1 °C, 5% CO2) with assay medium
- Main experiment: 60 ± 1 min exposure to dose groups

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg test item + 25 µL DPBS

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 replicate tissues

Test system

Details on study design:

Details of the test procedure used
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (EPI-200-SIT kit, Lot No.: 23380 Kit A)
- Conditions of exposure: 37 ± 1 °C, 5% CO2, 95% RH
- Washing: inserts gently rinsed with DPBS at least 15 times
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: 25 mg of test item per 1 mL of MTT medium and incubated for 60 minutes at 37 ± 1 °C
- Data evaluation: the following was calculated: The mean OD (optical density) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability(%) = (mean OD test item / positive control / mean OD negative control) x 100. For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated
- Description of evaluation criteria: The test item is considered to be irritant to skin in accordance with
regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%.
- Historical data positive control: Mean Viability: 4.0%; Rel. Standard Deviation: 2.0%
- Historical data negative control: Mean Absorption: 1.830; Rel. Standard Deviation: 0.376
- Acceptability of the assay: the results are acceptable if (1) tissue viability is meeting the acceptance criterion, i.e. if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8 (negative control). If (2) the relative tissue viability of the positive control is ≤ 20% (positive control). If (3) the SD of 3 identical replicates is < 18%. OD values should not be below historically established boundaries.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

>= 88.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The acceptance criteria were met

Any other information on results incl. tables

Result of the Test Item Mordant Red 11:

Name	Negative control			Positive Control			Test Material
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	2.021	1.928	1.971	0.105	0.107	0.113	2.107	1.411	1.728
	2.020	1.959	2.045	0.114	0.110	0.118	2.112	1.448	1.745
OD570 (blank-corrected)	1.978	1.885	1.928	0.062	0.064	0.070	2.064	1.368	1.685
	1.977	1.916	2.002	0.071	0.067	0.075	2.070	1.405	1.703
mean OD570 of the duplicates (blank-corrected)	1.978	1.900	1.965	0.067	0.066	0.073	2.067	1.387	1.694
total mean OD570of 3 replicate tissues (blank-corrected)	1.948*			0.068			1.716
SD OD570	0.042			0.004			0.340
relative tissue viability [%]	101.5	97.6	100.9	3.4	3.4	3.7	106.1	71.2	87.0
mean relative tissue viability [%]	100.0			3.5**			88.1
SD tissue viability [%]***	2.1			0.2			17.5
CV [% viabilities]	2.1			5.5			19.8

 

^*         Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^**        Mean relative tissue viability of the three positive control tissues is ≤ 20%.

^***       Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not irritant to skin according to UN GHS and EU CLP regulation.

Conclusions:

In this in vitro study under the given conditions the test item showed no irritation effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

The potential of the test item Mordant Red 11 to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. The test was performed according to OECD guideline 439 and in compliance to GLP.

In the present study the test item was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no non-specific reduction of MTT. The mixture of the test item with aqua dest. and isopropanol, respectively, showed colouring in the relvant wavelength range, therefore non-specific colour of additional viable tissues was determined. Since this was ≤ 5% (i.e. 1 %), no correction of the results was necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (88.1%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute optical density OD₅₇₀ of the three negative control tissues was ≥ 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 17.5%).