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Description of key information

Skin Irritation

REACH_not irritating | EpiDerm | OECD 439 | #key study#

Eye Irritation

REACH_not irritating | EpiOcular | OECD 492 | #key study#

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-22 to 2019-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
Version / remarks:
Version 07-Nov-2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Test system:
human skin model
Remarks:
EpiDerm
Source species:
human
Cell type:
non-transformed keratinocytes
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 skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Dose Groups
1. Negative control 30 µL Dulbecco’s phosphate buffered saline
2. Positive control 30 µL 5% sodium dodecyl sulfate solution
3. Test Item 30 µL (undiluted) / 25 mg + 25 µL Dulbecco’s phosphate buffered saline

Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
Post-treatment incubation: 42 ± 2 h
MTT incubation: 3h
Number of replicates:
The test was performed on a total of 3 tissues per dose group.
Details on study design:
The test was performed on EpiDerm, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (30 µL DPBS) and the positive control (30 µL 5% SDS), respectively. After 60 ± 1 min treatment period at 37°C (35 min) and room temperature (25 min) the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. The extracts are measured photometrically at 570 nm.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
48.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
57.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
72.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no 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 (1.883 [exp.1]; 1.798 [exp.2]; 2.121 [exp.3]). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.9% [exp.1]; 3.5% [exp.2]; 4.5% [exp.3]). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 17.9% [exp.1]; 0.5% - 6.5% [exp.2]; 0.3% - 7.7% [exp.3]).

The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 30 μL of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experiment 1: The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (48.9%) after 60 min treatment and 42 h post-incubation.

Experiment 2: The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (57.7%) after 60 min treatment and 42 h post-incubation.

Experiment 3: The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (72.7%) after 60 min treatment and 42 h post-incubation.

Result of the Test Item methyl-bis(2-arylpropyl)dihydro-heteropolycycle [exp.1]

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.991

1.766

1.859

0.134

0.134

0.132

1.293

0.852

0.674

2.017

1.787

1.878

0.134

0.131

0.143

1.325

0.840

0.673

Mean Absolute OD570

1.883****

0.135

0.943

OD570(Blank Corrected)

1.947

1.721

1.815

0.089

0.089

0.088

1.248

0.807

0.629

1.972

1.742

1.833

0.089

0.086

0.098

1.281

0.795

0.628

Mean OD570of the Duplicates
(Blank Corrected)

1.959

1.732

1.824

0.089

0.087

0.093

1.264

0.801

0.629

Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)

1.838*

0.090

0.898

SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)

0.115

0.003

0.329

Relative Tissue Viability [%]

106.6

94.2

99.2

4.8

4.8

5.1

68.8

43.6

34.2

Mean Relative Tissue Viability [%]

100.0

4.9**

48.9

SD of Relative Tissue Viability [%]***

6.2

0.2

17.9

CV [% Viabilities]

6.2

3.2

36.6

*          Blank-corrected mean OD570 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%

****     The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8.

Result of the Test Item methyl-bis(2-arylpropyl)dihydro-heteropolycycle [exp. 2]

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.663

1.848

1.876

0.099

0.098

0.113

0.952

1.081

1.086

1.671

1.871

1.859

0.099

0.099

0.116

1.021

1.085

1.107

Mean Absolute OD570

1.798****

0.104

1.055

OD570(Blank Corrected)

1.621

1.805

1.833

0.056

0.056

0.071

0.909

1.038

1.043

1.628

1.828

1.816

0.056

0.056

0.073

0.978

1.042

1.065

Mean OD570of the Duplicates
(Blank Corrected)

1.624

1.816

1.825

0.056

0.056

0.072

0.943

1.040

1.054

Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)

1.755*

0.061

1.012

SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)

0.113

0.009

0.060

Relative Tissue Viability [%]

92.5

103.5

104.0

3.2

3.2

4.1

53.7

59.2

60.0

Mean Relative Tissue Viability [%]

100.0

3.5**

57.7

SD of Relative Tissue Viability [%]***

6.5

0.5

3.4

CV [% Viabilities]

6.5

15.0

5.9

          Blank-corrected mean OD570 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%

****      The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8.

Result of the Test Item methyl-bis(2-arylpropyl)dihydro-heteropolycycle [exp. 3]

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.938

2.161

2.238

0.135

0.136

0.143

1.512

1.681

1.425

1.951

2.167

2.269

0.139

0.137

0.149

1.540

1.734

1.431

Mean Absolute OD570

2.121****

0.140

1.554

OD570(Blank Corrected)

1.893

2.115

2.192

0.089

0.090

0.098

1.467

1.635

1.380

1.906

2.121

2.224

0.094

0.091

0.103

1.494

1.688

1.386

Mean OD570of the Duplicates
(Blank Corrected)

1.899

2.118

2.208

0.092

0.091

0.100

1.481

1.662

1.383

Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)

2.075*

0.094

1.508

SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)

0.159

0.005

0.142

Relative Tissue Viability [%]

91.5

102.1

106.4

4.4

4.4

4.8

71.3

80.1

66.6

Mean Relative Tissue Viability [%]

100.0

4.5**

72.7

SD of Relative Tissue Viability [%]***

7.7

0.3

6.8

CV [% Viabilities]

7.7

5.7

9.4

*                 Blank-corrected mean OD570 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%.

****       The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8.

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

In the present study, in three independent experiments, methyl-bis(2-arylpropyl)dihydro-heteropolycycle

was applied topically to the EpiDermTM 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 mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 30 µL of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experiment 1:

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (48.9%) after 60 min treatment and 42 h post-incubation.

Experiment 2:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (57.7%) after 60 min treatment and 42 h post-incubation.

Experiment 3:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (72.7%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD570of the three negative control tissues was >= 0.8 and ≤ 2.8 (1.883 [exp.1]; 1.798 [exp.2]; 2.121 [exp.3]).

The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.9% [exp.1]; 3.5% [exp.2]; 4.5% [exp.3]).

Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 17.9% [exp.1]; 0.5% - 6.5% [exp.2]; 0.3% - 7.7% [exp.3]).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
2019-01-29 to 2019-01-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.
Version / remarks:
25 June 2018
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
Version / remarks:
22 July 2015
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
29 June 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Species:
human
Details on test animals or tissues and environmental conditions:
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate
3. Test Item 50 µL (undiluted)
The test was performed on a total of 2 tissues per dose group.
Duration of treatment / exposure:
incubation: 30 +/- 2 min.
Duration of post- treatment incubation (in vitro):
- post soak incubation: 12 +/- 2 min.
- post treatment incubation: 120 +/- 15 min.
- MTT incubation: 3h +/- 15 min.
Number of animals or in vitro replicates:
2
Details on study design:
The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.
In the present study the test item was applied topically to the EpiOcular tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min post-treatment period cytotoxic effects were determined via MTT reduction assay.
Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.
Irritation parameter:
other: mean relative tissue viability
Run / experiment:
1
Value:
104.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %
Other effects / acceptance of results:
Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.624 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 39.9 < 50% pass
Max. Difference of % Viability [%] 11.1 < 20% pass

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%. The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was >60% (104.6%).

Result of the Test Item Methyl-bis(2 -arylpropyl)dihydro-heteropolycycle

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

1.574

1.684

0.667

0.676

1.558

1.765

1.569

1.669

0.663

0.689

1.660

1.806

Mean Absolute OD570

1.624****

0.674

1.697

OD570(Blank Corrected)

1.531

1.640

0.624

0.632

1.514

1.721

1.525

1.626

0.619

0.646

1.617

1.762

Mean OD570of the Duplicates
(Blank Corrected)

1.528

1.633

0.621

0.639

1.566

1.742

Total Mean OD570of the 2 Replicate Tissues (Blank Corrected)

1.580*

0.630

1.654

SD of Mean OD570of the Duplicates (Blank Corrected)

0.074

0.012

0.125

Relative Tissue Viability [%]

96.7

103.3

39.3

40.4

99.1

110.2

Relative Tissue Viability
Difference [%]***

6.7

1.1

11.1

Mean Relative Tissue Viability [%]

100.0

39.9**

104.6

*              Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**             Mean relative tissue viability of the positive control is < 50%

***            Relative tissue viability difference of replicate tissues is < 20%

****        Mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.5       

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.
Executive summary:

In the present study the eye irritating potential of the test item was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular TM, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (104.6%).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin Irritation

The test substance did not produce skin irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Eye Irritation

The test substance did not produce eye irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

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