Registration Dossier

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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06-Jul-2012
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
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 OD570 nmof 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%).