Registration Dossier

Administrative data

Description of key information

Skin corrosion:

For this endpoint one key study is available. The skin corrosion potential was assessed with the in vitro Reconstructed Human Epidermis (RhE) test method (OECD 431, GLP). No corrosive effects were observed after 3 and 60 min treatment, leading to the conclusion that the test item is “non-corrosive“.

Skin irritation:

One key study is available in which the potential of the test item to induce skin irritation was analysed. An in vitro study was conducted using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum (OECD 439, GLP). After 60 min exposure and 42h post-incubation period, a mean relative tissue viability of 3.8 % was measured. As this is ≤ 50%, the substance is considered to be irritant.

Eye irritation:

The eye irritancy potential was investigated in the bovine corneal opacity and permeability assay (OECD 437, GLP). All 3 corneas treated with the test item showed an orange coloration and a very intense opacification of the issue. The mean in vitro irritation score (IVIS) was 357.21. Hence, the substance is considered to be irritant to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.12.2016-01.03.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
unchanged (no vehicle)
Details on test system:
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Pre-incubation: the inserts containing the tissues were set into the incubator at 37 ± 1°C, 5.0% CO2 / 95% air for 1 hour.
- For each experiment (“3 minutes” and “1 hour”), the plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2.
- MTT medium: the tissues were incubated with MTT medium for 3 h at 37 ± 1°C, 5.0% CO2 / 95% air.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg test item + 25 µL H2O
Duration of treatment / exposure:
3 and 60 minutes treatment
Number of replicates:
2 replicates for each treatment period (3 min and 60 min exposure time)
Details on study design:
Details of the test procedure
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (MatTek, EPI-200-SCT)
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: the tissue was gently rinsed about 20 times with PBS
- Number of tissue replicates used per test chemical and controls: 2
- MTT assay: incubation with 0.3 mL of MTT solution for 3 hours at 37 ± 1 °C, 5% CO2)

- Data evaluation:
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%) according to the following Prediction Model
In step 1:
< 50% after 3 min exposure: predicted as corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure: predicted as corrosive (a combin
ation of optional sub-categories 1B and 1C)
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: predicted as Non-Corrosive
In step 2:
< 25% after 3 min exposure: optional Sub-category 1A
≥ 25% after 3 min exposure: a combination of optional Sub-categories 1B and 1C

- Historical data negative control: Mean Absorption: 1.895 (3 min), 1.867 (1 h); Standard Deviation: 0.313 and 0.261, respectively
- Historical data positive control: Mean Viability: 6.1% (1 h); Standard Deviation: 1.99%

- The test meets acceptance criteria if:
- mean OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min. incubation time
Value:
>= 87.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not corrosive to skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour incubation time
Value:
>= 17.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: borderline result
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour incubation time
Value:
>= 20.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: repeated due to a borderline result
Remarks:
no corrosive effects
Other effects / acceptance of results:
The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was≥ 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (5.4% and 10.7%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (2.8% - 13.3%).

Results of the 3 min experiment:

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

2.006

1.759

1.723

1.516

0.098

0.180

2.021

1.738

1.839

1.490

0.097

0.182

2.055

1.751

1.883

1.525

0.099

0.189

OD570- Blank Corrected

1.963

1.715

1.680

1.473

0.055

0.137

1.977

1.695

1.796

1.447

0.054

0.139

2.011

1.708

1.840

1.482

0.056

0.146

Mean OD570of 3 Aliquots (blank corrected)

1.984

1.706

1.772

1.467

0.055

0.141

SD OD570 of 3 Aliquots

0.025

0.026

0.078

0.029

0.024

0.024

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.845

1.619

0.098

TODTT

-

1.608

-

SD OD570 of 2 Replicate Tissues

0.197

0.215

0.061

Mean Relative Tissue
Viability [%]

100.0

87.8

5.3

NSMTT-corrected mean relative tissue viability [%]

-

87.2

-

Coefficient Of Variation [%]

10.7

13.3

62.2

Results of the 60 min experiment:

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

1.627

1.683

0.346

0.275

0.155

0.110

1.601

1.733

0.339

0.278

0.155

0.111

1.641

1.721

0.339

0.270

0.156

0.113

OD570- Blank Corrected

1.582

1.638

0.302

0.230

0.110

0.065

1.556

1.688

0.294

0.233

0.110

0.066

1.596

1.676

0.294

0.226

0.111

0.068

Mean OD570of 3 Aliquots (blank corrected)

1.578

1.667

0.296

0.230

0.111

0.066

SD OD570 of 3 Aliquots

0.020

0.034

0.025

0.025

0.025

0.025

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.623

0.263

0.088

TODTT

-

0.289

-

SD OD570 of 2 Replicate Tissues

0.063

0.047

0.031

Mean Relative Tissue
Viability [%]

100.0

16.2

5.4

NSMTT-corrected mean relative tissue viability [%]

-

17.4

-

Coefficient Of Variation [%]

3.9

17.9

35.5

Because of the borderline result, a second experiment was conducted.

Results of the second 60 min experiment:

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

Absolute OD570

1.644

1.707

0.329

0.393

0.209

0.229

1.700

1.672

0.352

0.400

0.213

0.228

1.826

1.598

0.351

0.383

0.213

0.232

OD570- Blank Corrected

1.601

1.664

0.285

0.349

0.166

0.185

1.657

1.628

0.309

0.357

0.169

0.184

1.783

1.554

0.308

0.340

0.169

0.189

Mean OD570of 3 Aliquots (blank corrected)

1.680

1.616

0.301

0.349

0.168

0.186

SD OD570 of 3 Aliquots

0.093

0.055

0.027

0.025

0.024

0.024

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.648

0.325

0.177

TODTT

-

0.351

-

SD OD570 of 2 Replicate Tissues

0.046

0.034

0.013

Mean Relative Tissue
Viability [%]

100.0

19.7

10.7

NSMTT-corrected mean relative tissue viability [%]

-

20.9

-

Coefficient Of Variation [%]

2.8

10.4

7.2

Conclusions:
The test item showed no corrosive effects. The test item is classified as “non-corrosive“.
Executive summary:

In an in vitro study, the skin corrosion potential of the test item was assessed with the Reconstructed Human Epidermis (RhE)

test method according to OECD 431 and EU-Method B.40 and in compliance to GLP.

The test item was applied topically and cytotoxic effects to the stratum corneum after a short time exposure were determined. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

The test item showed non-specific MTT-reducing (NSMTT) potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT. After 3 min treatment, NSMTT was 0.6%, after 60 min treatment, NSMTT was -1.2%. The results were corrected accordingly. Moreover, the test item showed no colouring potential after mixture with aqua dest. but colouring was detected after mixture with isopropanol. Since there was no relevant absorption in the range of 570 ± 30 nm, NSCliving(non-specific colour of additional viable tissues) was not determined.

The corrosivity potential was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%). According to the Prediction Model: a mean relative tissue viability of ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure conclude to a non-corrosive effect of the test item. Due to a borderline result (17.4%) the 60 min treatment period was repeated. The mean relative tissues viability (% of negative control) was 87.2 % (NSMTT-redcuced) after 3 min treatment and was 17.4 % and 20.9% after 60 min treatment.

According to the Prediction Model, the test item showed no corrosive effects.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-08 to 2016-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 July 2012
GLP compliance:
yes (incl. certificate)
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
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.
Vehicle:
other: DPBS
Remarks:
to improve the contact between the powder 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 cm.
- Pre-incubation: 60 ± 5 minutes, then transferred into new wells for 18 ± 3 h in the incubator (37 ± 1 °C, 5% CO2) in the upper wells.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Negative control: 30 µL DPBS
Positive control: 30 µL 5% SDS solution
Test Item: 25 mg + 25 µL DPBS
Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 2 hours
Number of replicates:
3
Details on study design:
Details of the test procedure used:
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (EPI-200-SIT)
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: inserts gently rinsed with DPBS
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation of 25 mg of test item per 1 mL MTT medium for 60 minutes at 37 ± 1 °C
- Data evaluation: the following was calculated: The mean OD of the three negative control tissues was calculated after blank correction. The mean of the photometric absorbance of the negative control is set to 100%. 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:
- GHS Cat 2 according to UN GHS is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control
- GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%
- Historical data positive control: Mean Viability: 3.9%; Rel. Standard Deviation: 4.4%
- Historical data negative control: Mean Absorption: 1.831; Rel. Standard Deviation: 0.357;

The test meets acceptance criteria if:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Single test with three tissues
Value:
3.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The controls confirmed the validity of the study:
- the mean absolute 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% - 2.1%).

Results of the pre-tests:

- No correction for the non-specific MTT reduction was required because the test outcome is positive.

- No colouring was detectable in the unaided eye assessment. However, since the test outcome is positive, no correction procedure for determination of NSC was necessary.

Test results main experiment:

Name

Negative Control

Positive Control

Test item

Tissue

1

2

3

1

2

3

1

2

3

absolute OD570

2.021

1.928

1.971

0.105

0.107

0.113

0.105

0.123

0.118

2.020

1.959

2.045

0.144

0.110

0.118

0.106

0.126

0.123

OD570(blank-corrected)

1.978

1.885

1.928

0.062

0.064

0.070

0.062

0.080

0.075

1.977

1.916

2.002

0.071

0.067

0.075

0.063

0.083

0.080

mean OD570of the duplicates (blank-corrected)

1.978

1.900

1.965

0.067

0.066

0.073

0.063

0.082

0.078

total mean OD570of 3 replicate tissues (blank-corrected)

1.948

0.068

0.074

SD OD570

0.042

0.004

0.010

relative tissue viability [%]

101.5

97.6

100.9

3.4

3.4

3.7

3.2

4.2

4.0

mean relative tissue viability [%]

100.0

3.5

3.8

SD tissue viability [%]

2.1

0.2

0.5

CV [% viabilities]

2.1

5.5

13.4

Conclusions:
The mean relative tissue viability (% negative control) was 3.8 % after 60 min treatment and 42h post-incubation.Therefore it is concluded that the test item showed skin irritant effects.
Executive summary:

The potential of the test item 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 TG 439 and in compliance to GLP.

The test item was applied topically to the EpiDerm tissue for 60 min exposure 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 mean relative tissue viability (% negative control) was 3.8 % (≤ 50%) after 60 min treatment and 42 h post-incubation. The test item showed MTT-reducing capability. However, since the substance was classified as “Irritant” in the main experiment, no correction procedures for determination of NSMTT were necessary. The test item showed colouring potential after mixture with isopropanol. Nevertheless, since the substance was classified as “Irritant” in the main experiment, no correction procedures for determination of NSC were necessary. It can be concluded that the test item showed irritant effects.

The controls confirmed the validity of the study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.05.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
purity: 100%
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Freshly isolated bovine cornea obtained as a by-product from animals freshly slaughtered
- Source: A. Moksel AG, Buchloe, Germany
Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 L
Duration of treatment / exposure:
- 4 hours ± 5 minutes at 32 ± 1 °C, washed at least three times with MEM (containing phenol red), finally rinsed with complete RPMI (without phenol red)
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
- After the illuminance measurement was performed the corneas were incubated for 90 minutes at 32 ± 1 °C
Number of animals or in vitro replicates:
3 corneas/group
Details on study design:
Preparation of the Corneas:
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS
containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2
to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the
corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring
of the posterior chamber, they had been visually examined for defects and any defective cornea had
been discarded. The anterior chamber was then positioned on top of the cornea and tightened with
screws.
The chambers of the corneal holder were then filled with RPMI (without phenol red) containing
1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The
corneas were incubated for one hour at 32 ± 1 °C.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
An initial measurement was performed on each of the corneas using the opacitometer.
Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as
negative-control corneas.
The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.
The medium was removed from the anterior chamber and replaced with the test item or control.
750 L of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed and the
epithelium washed at least three times with MEM (containing phenol red).
Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder.
The posterior chamber was refilled with fresh complete RPMI.
1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1°C.
Then the medium from theposterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneas / 4 h incubation time
Value:
357.21
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Deviations:
Deviations did not influence the quality or integrity of the present study

In Vitro Irritation Score

Cornea no. Test item Corrected opacity Corrected OD490 value IVIS
1 Negative control -0.04 0.006 0.67
2 0.15 0.015
3 1.35 0.015
MV 0.49 0.012
4 Positive control 76.72 0.951 97.7
5 82.82 0.932
6 88.91 1.094
MV 82.82 0.992
7 Test item 276.79 5.528 357.21
8 334.61 3.203
9 267.57 4.113
MV 292.99 4.281
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
A mean in vitro irritation score of 357.21 was calculated.
According to the evaluation criteria the test item Ingrain Blue 2.2 is classified into UN GHS Category 1.
Executive summary:

The eye irritancy potential of Ingrain Blue 2.2 was investigated in the bovine corneal opacity and permeability assay.

The test was perforemd in accordance to the OECD TG 437 and in compliance to GLP.

All 3 corneas treated with Ingrain Blue 2.2 showed a very intense opacification and an orange discoloration of the tissue.

The following mean in vitro irritation score was calculated: 357.21

It cannot be excluded that the high opacity score may be related to the orange discolouration of the tissue but due to the observed intense opacity of the treated corneas and the high rate of the score, an eye irritancy potential of the test item is assumed.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

A mean in vitro irritation score of 357.21 was calculated.

According to the evaluation criteria the test item Ingrain Blue 2.2 is classified into UN GHS Category 1.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion:

In an in vitro study the skin corrosion potential was assessed with the Reconstructed Human Epidermis (RhE) Test Method (OECD 431; EU-Method B.40; GLP). The corrosivity potential was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%). According to the Prediction Model: a mean relative tissue viability of ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure conclude to a non-corrosive effect of the test item. The mean relative tissues viability (% of negative control) was 87.2 % (NSMTT-reduced) after 3 min treatment and 17.4% (first rep) and 20.9% (2nd rep) after 60 min treatment. Due to a borderline result (17.4%) the 60 min treatment period was repeated. It can be concluded that in this study the test item showed no corrosive effects.

Skin irritation:

The potential of the test item 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 (OECD 439, GLP). The test item showed irritant effects. The mean relative tissue viability (% negative control) was 3.8% after 60 min treatment and 42 h post-incubation. Since this ≤ 50 % it is concluded that the substance is skin irritant.

Eye irritation:

The eye irritancy potential was investigated in the bovine corneal opacity and permeability assay (OECD 437, GLP). All 3 corneas treated with Ingrain Blue 2.2 showed a very intense opacification and an orange discoloration of the tissue. The following mean in vitro irritation score was calculated: 357.21. It cannot be excluded that the high opacity score may be related to the orange discolouration of the tissue but due to the observed intense opacity of the treated corneas and the high rate of the score, an eye irritancy potential of the test item is assumed.

Justification for classification or non-classification

Skin corrosion:

The mean relative tissues viability (% of negative control) was 87.2% (NSMTT-reduced) after 3 min treatment and 17.4% and 20.9% after 60 min treatment. According to the Prediction Model: a mean relative tissue viability of ≥ 50% after 3 min exposure and ≥ 15% after 60 min exposure concludes to a non-corrosive effect of the test item. Based on these test results, the test item should be classified as “non-corrosive“ according to CLP (EC No 1272/2008).

Skin irritation:

The test item showed a mean relative tissue viability of 3.8%. The test item is considered irritant to the skin since the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control. Thus, based on the available data on skin irritation, the test item is classified as “irritant” in accordance with the criteria for classification according to Regulation (EC) 1272/2008 (CLP) and classified "Category 2".

Eye irritation:

A mean in vitro irritation score of 357.21 was calculated in the bovine corneal opacity and permeability assay. According to Regulation (EC) 1272/2008 (CLP) the test item is classified as "Category 1", causing serious eye damage.

It has to be concluded that according to Regulation (EC) 1272/2008 (CLP), that the test item is classified as “non-corrosive“ to skin, “irritant” to skin "Category 2", and classified into "Category 1" for eye irritation.