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Description of key information

The substance was tested in the EPISKIN™ Reconstructed Human Epidermis Model using triplicate tissues during 15 minutes. The relative mean viability of the test substance treated tissues was 100.7 %. Therefore it can be concluded that the substance is not irritant to the skin.

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. The relative mean viability of the test substance treated tissues was 98.0 % for the 3 minutes exposure and 101.5% for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test with exposure to 50 µL of the test item, the negative control (deionised water) or the positive control. Irritating effects were not observed following incubation with the substance. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (92.1%).

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:
16 August 2017 to 18 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008,
Version / remarks:
laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: EpiDerm™ Human Skin Mode
Cell source:
other: EpiDerm™ Human Skin Mode
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Human Skin Mode (MatTek)
- Tissue batch number(s): 25837
- Delivery date: 15 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ˚C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ˚C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsed under a constant soft stream of DPBS and blotted dry with a tissue

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nM

NUMBER OF REPLICATE TISSUES: 2/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50% (H314 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (H314 1B or 1C)
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15% (not classified as corrosive)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.05 mL as such

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8.0 N Potassium Hydroxide
Duration of treatment / exposure:
3 min and 60 min
Duration of post-treatment incubation (if applicable):
The plates was incubated (37 ˚C, 5% CO2) for 3 hours in presence of MTT. Thereafter tissues were placed in isopropanol for MTT extraction, which was measured in triplicate samples as optical density at 570 nm.
Number of replicates:
2/treatment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
A 3 minutes exposure
Value:
98
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
B 60 minutes exposure
Value:
101.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
The results were in agreement with the quality criteria:

Negative Control
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD 570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

 

 

Exposure time

Mean OD570

Relative mean viability

Negative control

3 min

1.586

100%

 

60 min

1.683

100%

Test substance

3 min

1.554

98.0%

 

60 min

1.708

101.5%

Positive control

3 min

0.064

4.0%

 

60 min

0.058

3.4%

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is considered non-corrosive
Executive summary:

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. The relative mean viability of the test substance treated tissues was 98.0 % for the 3 minutes exposure and 101.5% for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2017 to 16 October 2017
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)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Cell source:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s):17-EKIN-041 (0.38cm2)
- Delivery date: 10 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ˚C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ˚C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS:rinsed using a wash bottle containing DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nM

NUMBER OF REPLICATE TISSUES: 3/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Relative mean tissue viability is ≤50% --> irritant (H315)
- Relative mean tissue viability is >50% --> non-irritant (not classified)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg as such ( (26.3 mg/cm2)

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL SDS (5%)
Duration of treatment / exposure:
15 min at room temperature (thereafter rinsed with DPBS)
Duration of post-treatment incubation (if applicable):
The plates were incubated (37 ˚C, 5% CO2) for 42 hours thereafter shaken to homogenize. Thereafter incubated for 3 hours in presence of MTT. The epidermis and the collagen matrix were separated and immersed in acidified isopropanol. The tissues were stored in a refrigirator for 6 days at 1-10 ˚C for MTT extraction, which was measured in triplicate samples as optical density at 570 nm.
Number of replicates:
3/treatment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minutes exposure
Value:
100.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The results were in agreement with the quality criteria:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

   

 

Exposure time

Mean OD570

Relative mean viability

SD

Negative control

15 min

0.873

100%

7.9%

Test substance

15 min

0.879

100.7%

5 %

Positive control

15 min

0.204

23.4%

5 %

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is considered non-irrirant
Executive summary:

The substance was tested in the EPISKIN™ Reconstructed Human Epidermis Model using triplicate tissues during 15 minutes. The relative mean viability of the test substance treated tissues was 100.7 %. Therefore it can be concluded that the substance is not irritant to the skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 July 2017 to 31 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 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:
MatTek Corporation Protocol: 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; 29 June 2015.
GLP compliance:
yes (incl. QA statement)
Species:
other: human keratinocytes
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).

Lot No.: 27002
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free D-PBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 uL
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
12 minutes in Assay Medium (to remove any test item absorbed into the tissue) and 120 minutes in Assay Mediumat 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
Number of animals or in vitro replicates:
2 replicates per treatment (except for blank)
Details on study design:
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues (3 times) with Ca++Mg++-free DPBS (brought to room temperature).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that no isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Irritation parameter:
other: viability %
Value:
92.1
Negative controls validity:
valid
Positive controls validity:
valid

Dose Group

Ab-sorbance
Well 1
(Tissue 1/2)

Ab-sorbance
Well 2 (Tissue 1/2)

Mean Absor-bance (Tissue 1/2)

Mean Absorbance* Tissue 1 and 2

Mean Absorbance of
2 Tissues*

Rel. Absorbance [%]
Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Mean Rel. Absorbance

[%]***

Blank

0.038

0.038

0.038

0.000

 

99.1

 

 

Negative Control

1.674

1.753

1.714

1.676

1.691

100.9

1.8

100.0

1.735

1.753

1.744

1.706

36.8

Positive Control

0.626

0.693

0.660

0.622

0.617

36.2

0.6

36.5

0.647

0.652

0.649

0.611

92.6

Test Item

1.555

1.651

1.603

1.565

1.558

91.7

0.9

92.1

1.581

1.595

1.588

1.550

99.1

*                            Mean of two replicate wells after blank correction
**                          Relative absorbance [rounded values]: 100 * (absorbace test item/positive control/negative control)/absorbance negative control

***                        Mean relative absorbance [rounded values]: 100 * (mean absorbace test item/positive control/negative control)/mean absorbance negative control

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not irritating to the eyes
Executive summary:

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test. The substance did not prove to be an MTT reducer and color interference was excluded.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the substance. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (92.1%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance does not possess any eye irritating potential.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not indicated
-- Storage, temperature and transport conditions of ocular tissue: placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL), transported over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: not indicated
- indication of any existing defects or lesions in ocular tissue samples: checked and only undamaged tissue used
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL: 0.75 mL as such
Duration of treatment / exposure:
10 minutes at 32 ± 1 ºC
Duration of post- treatment incubation (in vitro):
post treatment opacity measurement immediately
permeality measurement after exposure to fluorescein for 120 min at 32 ± 1 ºC
Details on study design:
QUALITY CHECK OF THE ISOLATED CORNEAS:based on visual examination and pre-treatment opacity check

NUMBER OF REPLICATES: 3/treatment

TREATMENT METHOD:closed chamber

REMOVAL OF TEST SUBSTANCE: 3 rinses with EMEM containing phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity and Corneal permeability

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage
Irritation parameter:
in vitro irritation score
Value:
1.3
Negative controls validity:
valid
Positive controls validity:
not valid
Remarks:
see below
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The positive control group had an overall IVIS of 68.4 which was higher than the criteria range set for an acceptable test. However, it was decided that the results were considered acceptable as the positive control group still provided its’ intended function. The result of the test item gave a score that was consistent with a non-irritant, so therefore the corneas were considered not to have been compromised.
This deviation was considered to have not affected the integrity or validity of the study.

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

10

3

 

0.054

 

 

11

3

 

0.020

 

 

12

2

 

0.092

 

 

 

2.7

 

0.055

 

3.5

Positive Control

13

33

30.3

2.630

2.575

 

14

34

31.3

3.025

2.970

 

15

28

25.3

2.385

2.330

 

 

 

29.0

 

2.625

68.4

Test Item

16

5

2.3

0.035

0.000

 

17

4

1.3

0.021

0.000

 

18

3

0.3

0.018

0.000

 

 

 

1.3

 

0.000

1.3


OD= Optical density         

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the test the substance is considered not severely damaging to the eyes
Executive summary:

In a BCOP assay bovine corneas were exposed to the substance and assessed for opacity and permeability. No effects of the substance on opacity readings and permeability was observed. The calculation of the In Vitro Irritancy Score (1.3) showed that the substance is not severely damaging to the eyes.

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

Based on the available information no classification for skin- and eye irritation is necessary according to Regulation (EC) No 1272/2008 (CLP).