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Administrative data

Description of key information

Based on the study result obtained from a reliable in vitro test according to OECD 431, the substance is not considered to be corrosive.

Based on the study result obtained from a reliable in vitro test according to OECD 439, the substance will be classified according to CLP as Category 2 (H315: Causes skin irritation).

For eye corrosion/irritation two in vitro tests according to OECD 437 are available. Both tests show no predictable results based on the IVIS Score. In the study used as key (2018), a histopathology evaluation of corneas from eyes exposed to the test item was conducted and gave a score exceeding that of the positive control corneas. Thus, the substance will be classified according to CLP as Category 1 (H318: Causes serious eye damage).

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:
04-01-2017 to 02-02-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
EC Directive 2000/33/EC, OJ L136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40 and EC regulation No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation No 1907/2006 of the European Parliament and of the Council on REACH, 1st ATP, section B40.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted July 29, 2016
Qualifier:
according to
Guideline:
other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”
Version / remarks:
07 November 2014
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
EpiDerm™ tissues models
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Human Skin Model Test with EpiDerm™ tissues models
- Tissue batch number(s): EpiDerm™ Kit Lot No.: 23390
- Delivery date: 31 Jan 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
for the 3 ± 0.5 minutes exposure periods: room temperature
for the 60 ± 5 minutes exposure period plates were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2)

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period the tissues were gently rinsed using a wash bottle or multipipette containing DPBS (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution (300 µL) was added to each well
- Incubation time: 3 hours
The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1) at 570 nm (OD570) without reference filter.

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:
50 µL (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface.
Duration of treatment / exposure:
3 minutes and 60 minutes
Number of replicates:
2 Duplicates per Exposure Interval:
2 Duplicates / 3 min
2 Duplicates / 60 min
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
66.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Interpretation of results:
GHS criteria not met
Conclusions:
Both values 56.0% (3 min exposure) and 66.6% (60 min exposure) did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.
Under the experimental conditions according to OECD guideline 431, the test item is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

Both values 56.0% (3 min exposure) and 66.6% (60 min exposure) did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Under the experimental conditions, the test item is non corrosive to skin according to EU CLP and UN GHS.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-01-2017 to 10-02-2017
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:
Original Guideline adopted July 28, 2015, and as described in detail in the INVITTOX Protocol: SKINETHICTM SKIN IRRITATION TEST–42bis, January 2011.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.
Principles of method if other than guideline:
UN GHS (published 2003, last (6th) revision 2015)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
EpiSkin™
Source species:
other: Normal Human-Derived Epidermal Keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 17-EKIN-006
- Delivery date: 07 February 2017
- Date of initiation of testing: Day of receipt

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37+/- 1.5 degrees C
- Temperature of post-treatment incubation (if applicable): 37+/- 1.5 degrees C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After treatment, the inserts were immediately removed from 12-well plate and gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking inserts and blotting bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium and incubated for approximately 42 hours at 37 +/- 1.5 degrees C and 5 +/- 0.5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hour
- Spectrophotometer: OD was read in a microplate reader
- Filter bandwidth: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: mean OD of three tissues is >=0.6 <=1.5

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

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

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%, 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 [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%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL (26.3 µL/cm2) of the undiluted test item were applied to each of the triplicate tissues
Duration of treatment / exposure:
15 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 samples
Value:
15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
After treatment with the test item, the corrected mean relative absorbance value was reduced to 15.0%. This value is below the threshold for irritancy of <= 50%. Therefore, the test item is considered to possess an irritant potential.
In this study and under the experimental conditions, the test item is irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

In this study using OECD 439 guidelines and under the experimental conditions, the test item is irritant to skin according to UN GHS and EU CLP regulation.

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:
2018-05-31 to 2018-05-31
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
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- source: abattoir
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: no; those 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(s) applied (volume or weight with unit): 0.75mL same amount for test item, positive and negative control
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
two hours
Number of animals or in vitro replicates:
3 per treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60minutes.

At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
3 per treatment

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
Ethanol (purity: >99.8% )

APPLICATION DOSE AND EXPOSURE TIME
0.75mL for 10 minutes

POST-INCUBATION PERIOD: yes, 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
3 times washing with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein:
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability Determinations:
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. A dilution was performed on the positive controls and the optical density was re-measured.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
negative control: IVIS = opacity value + (15 x OD492 value)
positive control and the test item: IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD492 value)

DECISION CRITERIA:
IVIS <= 3: No category. Not requiring classification to UN GHS or EU CLP
IVIS > 3; <= 55: No prediction of eye irritation can be made
IVIS > 55: classified as Category 1 UN GHS or EU CLP Causes serious eye damage

HISTOPATHOLOGY: Yes
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
45.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

 Treatment 

Cornea

Number

  Opacity                 
  Permeability (OD492)     
  In Vitro Irritancy Score
 Pre-Treatment  Post-Treatment  Post- Incubation

 Post-Incubation -

Pre-Treatment

Corrected

Value

 

Corrected

Value 

 


Negative Control

 

 

 

 

 

 1

 

 

 5

 6

 -

 0.033

 -

 -

 2

 

 

 2

 1

2

 -

0.004 

 -

 -

 3

 

 

 2

 2

 2

 0

 -

 0.007

 -

 -

 

 -

 

 -

 -

 -

 0.3*

 -

 0.015¿

 -

 0.6

Positive Control      

 4

 

 

 4

 27

 32

 28  27.7  2.435  2.420  -
 5     3  26  28  25  24.7  1.765  1.750  -
 6     2  31  32  30  29.7  1.790  1.775  -
    -  -  -  -  -  27.3•  -  1.982•  57.1
Test Item           7     3  32  48  45  44.7  0.116  0.101  -
 9     6  32  48  42  41.7  0.230  0.215  -
 10     4  37  48  44  43.7  0.100  0.085  -
    -  -  -  -  -  43.3•  -  0.134•  45.3

OD = Optical density * = Mean of the post-incubation  - pre-treatment values ¿ = Mean permeability   • = Mean corrected value

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
With an IVIS Score of 45.3 no prediction of eye irritation can be made. The histopathology evaluation of corneas from eyes exposed to the test item gave a score exceeding that of the positive control corneas.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the study result obtained from a reliable in vitro test according to OECD 431, the substance is not considered to be corrosive.

Based on the study result obtained from a reliable in vitro test according to OECD 439, the substance will be classified according to CLP as Category 2 (H315: Causes skin irritation).

For eye corrosion/irritation two in vitro tests according to OECD 437 are available. Both tests show no predictable results based on the IVIS Score. In the study used as key (2018), a histopathology evaluation of corneas from eyes exposed to the test item was conducted and gave a score exceeding that of the positive control corneas. Thus, the substance will be classified according to CLP as Category 1 (H318: Causes serious eye damage).