Registration Dossier

Administrative data

Description of key information

Skin

In an in vitro skin irritation study (RHE) according to OECD Guideline 439, a cell viability of 11.5 % was determined. The test substance showed skin irritating properties.

In an in vitro skin corrosion study (RHE) according to OECD Guideline 431, a cell viability of 103.0 % after 3 min exposure and 65.8 % after 60 min exposure was determined. The test item showed no skin corrosive properties.

Eye

in an in vitro eye irritation study (BCOP) according to OECD Guideline 437, an IVIS of 1.42 was calculated. The test substance did not show eye irritating properties.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2017-05-05 to 2017-06-30
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:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Demanded by the OECD Guideline 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT, EpiDerm™ tissue (MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue Lot number: 25825
- Delivery date: 2017-06-27
- Date of initiation of testing: 2017-06-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: 35 min at 37 ± 1.5 °C, follwed by 25 min at room temperature
- Temperature of post-treatment incubation: ~ 41 h at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h at 37 ± 1.5 °C
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
30 μL of the test item were added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. Since the test item did not dye water an additional test (step 2) with viable tissues (but without MTT addition) was not necessary to be performed.

The test item was further evaluated for its potential to interfere with the MTT assay. To test if a test item directly reduces MTT, 30 μL of the test item was added to 1 mL of the MTTsolution (1 mg/mL) and the mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control. Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean relative tissue viability of three individual tissues is reduced ≤ 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
41 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
11.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

 

Mean absorbance

Mean rel. Absorbance (%)

Rel. S.D (%)

Negative Control

1.670

100.0

4.6

Positive Control

0.055

3.3

0.6

Test substance

0.192

11.5

6.4

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In an in vitro skin irritation study (RHE) according to OECD Guideline 439, a cell viability of 11.5 % was determined. The test item showed skin irritating properties.
Executive summary:

In an in vitro study (RHE) according to OECD Guideline 439, the skin irritating potential of the test item was assessed. 30 µL of the neat test item, the positive control (5 % SDS solution), or the negative control (DPBS) were dispensed directly atop the EpiDerm™ tissue (triplicates) and incubated for 1 h at 37 ± 1.5 °C. After the incubation phase, all tissues were each rinsed from the test item. Further, the tissues were incubated for another 41 h at 37 ± 1.5 °C. After the post-incubation phase, cell viability was determined via MTT-assay. Concurrent positive and negative controls were valid. A cell viability of 11.5 % was determined after incubation with the test item. Based on the prediction model, the test item has to be considered as skin irritating.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2017-09-12 to 2017-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion, RHE)
Version / remarks:
2008-05-30
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”.
Version / remarks:
2014-11-07
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Demanded by the OECD Guideline 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues model (EPI-200 Kit)
- Tissue lot number: 25847
- Delivery date: 2017-10-04

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min exposure at RT; 60 min exposure at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times).
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)).
- Wavelength: 570 nm (OD570)
- Filter: without reference filter.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- No. of replicates: 2
- Method of calculation used:
True viability = Viability of treated tissue – Interference from test chemical =
ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, 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%.
- The test substance is considered to be non-corrosive to skin if 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 %.]
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
3 min or 60 min
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
103
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
68.8
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

 Table 3: Results after test item exposure and controls

Dose Group

Exposure Interval
[min]

Mean Ab-sorbance (OD) of 2 Tissues

Mean Rel. Absorbance
[%]*

CV [%]

Corrected Rel. Absorbance [%]**

Blank

3 min

 

 

 

 

Neg. Control (nc)

1.247

100.0

6.5

103.0

Pos. Control (pc)

0.218

17.4

15.8

Test Item

1.283

102.9

3.1

Neg. Control FK

0.080

6.4

12.2

Test Item FK

0.079

6.3

1.9

Blank

60 min

 

 

 

 

Neg. Control (nc)

1.226

100.0

4.6

65.8

Pos. Control (pc)

0.040

3.3

7.6

Test Item

0.810

66.0

10.4

Neg. Control FK

0.077

6.3

7.5

Test Item FK

0.080

6.5

0.9

 

* Mean Relative Absorbance =

(Mean Absorbance test item;pc;nc / Mean Absorbance nc) x 100

 

** Corrected Relative Viability (%) =

[Mean OD test item – (OD test item FK – OD nc fk) / Mean OD nc] x 100

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin corrosion study (RHE) according to OECD Guideline 431, a cell viability of 103.0 % after 3 min exposure and 65.8 % after 60 min exposure was determined. The test item is not consiedered to be corrosive.
Executive summary:

In an in vitro study (RHE) according to OECD Guideline 431, the skin corrosive potential of the test item was assessed. 50 µL of the neat test item, the positive control (8N Potassium hydroxide), or the negative control (deionised water) were dispensed directly atop the EpiDerm™ tissue (duplicates) and incubated for 3 min at RT or for 60 min at 37 ± 1.5 °C. After the incubation phase, all tissues were each rinsed from the test item. After the exposure, cell viability was determined via MTT-assay. Concurrent positive and negative controls were valid. A cell viability of 103.0 % after 3 min and 65.8 % after 60 min was determined after incubation with the test item. Based on the prediction model, the test item has to be considered as non-skin corrosive.

.

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:
from 2017-05-05 to 2017-05-05
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:
2013-07
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 months old
- Storage temperature: ambient temperature
- Transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin).
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3 corneas per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first. Care was taken to assure no air bubbles were present within the compartments. The corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. At the end of the incubation period, the basal opacity was determined (t0). Each corneae with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 min

POST-INCUBATION PERIOD: yes (2 hours)

REMOVAL OF TEST SUBSTANCE
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: OP_KIT opacitometer (changes in the light transmission passing through the cornae)
- Corneal permeability: Microtiter plate reader (OD490, passage of sodium fluorescein dye)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3 No Category (UN GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Category 1 (UN GHS)
Irritation parameter:
in vitro irritation score
Value:
1.42
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Table 1: Results after 10 Minutes Treatment Time

Test Group

Opacity value =

Difference (t130 –t0)

of Opacity

Permeability at

490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy score

 

 

Mean

 

Mean

 

 

 

Neg. Control

0

0.00

0.070

0.073

1.05

1.09

Not categorized

0

0.062

0.93

0

0.086

1.29

Pos. Control

57.00*

1.065*

72.98

83.62

Category I

65.00*

1.433*

86.50

69.00*

1.491*

91.37

Test item

2.00*

-0.020*

1.71

1.42

Not categorized

2.00*

-0.014*

1.80

1.00*

-0.017*

0.75

*corrected values

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the OECD 437 study, the test item does not show eye irritating properties..
Executive summary:

In an in vitro study according to OECD TG 437, the corneal damage potential of the test item was assessed. After a first opacity measurement of the fresh bovine corneae (t0), 0.75 mL of the neat test item, the positive control (2-Ethoxyethanol), and the negative control (saline) were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. The test item was tested undiluted. Relative to the negative control, the test item caused no increase of the corneal opacity and permeability. The calculated mean IVIS was 1.42 (threshold for no categorisation: IVIS 3). The required acceptability criteria were met. The test item is not considered to be classified as eye irritating.

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

Additional information

Skin

In an in vitro study (RHE) according to OECD Guideline 439 (2017), the skin irritating potential of the test substance was assessed. 30 µL of the neat test item, the positive control (5 % SDS solution), or the negative control (DPBS) were dispensed directly atop the EpiDerm™ tissue (triplicates) and incubated for 1 h at 37 ± 1.5 °C. After the incubation phase, all tissues were each rinsed. Further, the tissues were incubated for another 41 h at 37 ± 1.5 °C. After the post-incubation phase, cell viability was determined via MTT-assay. Concurrent positive and negative controls were valid. A cell viability of 11.5 % was determined after incubation with the test substance. Based on the prediction model, the test item has to be considered as skin irritating.

In an in vitro study (RHE) according to OECD Guideline 431, the skin corrosive potential of the test item was assessed. 50 µL of the neat test item, the positive control (8N Potassium hydroxide), or the negative control (deionised water) were dispensed directly atop the EpiDerm™ tissue (duplicates) and incubated for 3 min at RT or for 60 min at 37 ± 1.5 °C. After the incubation phase, all tissues were each rinsed from the test item. After the exposure, cell viability was determined via MTT-assay. Concurrent positive and negative controls were valid. A cell viability of 103.0 % after 3 min and 65.8 % after 60 min was determined after incubation with the test item. Based on the prediction model, the test item has to be considered as non-skin corrosive.

Eye

In an in vitro study according to OECD TG 437 (BCOP) (2017), the corneal damage potential of the test item was assessed. After a first opacity measurement of the fresh bovine corneae (t0), 0.75 mL of the neat test item, the positive control (2-Ethoxyethanol), and the negative control (saline) were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. The test item was tested undiluted. Relative to the negative control, the test item caused no increase of the corneal opacity and permeability. The calculated mean IVIS was 1.42 (threshold for no categorisation: IVIS 3). The required acceptability criteria were met. The test item is not considered to be classified as eye irritating.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on this data, the substance is not considered to be classified as eye irritating but has to be classified as skin irritating (Cat. 2, H315) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.