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Administrative data

Description of key information

Skin: The test item was found to be irritating to skin.

Eyes: The test item showed no eye irriation/corrosion potential.

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:
weight of evidence
Study period:
20 September 2018 to 31 October 2018
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:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.40 Bis: In Vitro Skin Corrosion: Human Skin Model Test
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
The EPISKIN-model is a 3-dimensional human epidermis model obtained after a 13-days culture period of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed human epidermis units are contained in the EpiSkin(TM)SM kits, supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 – LYON Cedex 07, France
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN model
- Tissue batch number: 18-EKIN-038

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
The skin units were rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Linearity range of spectrophotometer: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is not considered to be corrosive to skin if the mean tissue viability is ≥ 35 % after 4 hours exposure.
- The test substance is considered to be corrosive to skin (Cat 1 A) if the mean tissue viability is < 35 % after 3 min exposure.
- The test substance is considered to be corrosive to skin (Cat 1 B or C) if the mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure OR ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure
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:
4 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The mean OD value of the two negative control tissues was 0.763.
The positive control result showed 7 % viability.
The difference of viability between the two tissue replicates was between 2.7 % to 9.3 %.
All validity criteria were within acceptable limits. Therefore, the study can be considered as valid.

Table 1: OD values and viability percentages of the controls:

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
NaCl (9 g/L saline)

1

0.799

105

9.3

2

0.728

95

mean

0.763

100

 

Positive Control:
Glacial acetic acid

1

0.063

8

2.7

2

0.043

6

mean

0.053

7

 

 

Table 2: OD values and viability percentages of the test item (including corrected values):

 

Optical Density (OD)

Viability (%)

Δ%

Test substance

1

0.758

99

7.1

2

0.812

106

mean

0.785

103

 

 

Interpretation of results:
other: non-corrosive, further information necessary to decide on irritation potential and final classification
Conclusions:
The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.
Executive summary:

The study was conducted to determine the skin corrosion potential of the test item. Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours (±10 min) at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with 5±1 % CO2 in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 103 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential. This result can be used together with further information on the irritative nature of the substance to decide on its final classification.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 August 2018 to 18 September 2018
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
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
The EPISKIN-model is a 3-dimensional human epidermis model obtained after a 13-days culture period of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed human epidermis units are contained in the EpiSkin(TM)SM kits, supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 – LYON Cedex 07, France
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
The EpiSkin units were rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm (± 10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752)

PREDICTION MODEL / DECISION CRITERIA
Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number: 18-EKIN-033

NUMBER OF REPLICATE TISSUES: 3




Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL 1 x PBS

POSITIVE CONTROL
- Amount applied: 10 μL positive control (SDS 5 % aq.)
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
2 days
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
23
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: OD values and viability percentages of the controls:

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.222

97

2

1.199

95

3

1.352

108

mean

1.258

100

standard deviation (SD)

6.57

Positive Control:
SDS (5 % aq.)

1

0.162

13

2

0.165

13

3

0.162

13

mean

0.163

13

standard deviation (SD)

0.13

 

Table 2:OD values and viability percentages of the test item:

Substance

Optical Density (OD)

Viability (%)

Test Item

1

0.228

18

2

0.352

28

3

0.286

23

mean

0.289

23

standard deviation (SD)

4.90

 

Interpretation of results:
other: This test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2
Conclusions:
The results obtained from this in vitro skin irritation test ndicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information is necessary.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN modelin vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. The epidermis units were then incubated at 37 ± 1 °C for 42 hours (± 1h). 1xPBS served as negatice control, sodium dodecyl sulphate as positive control. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37 ± 1 °C. The resulting formazan was quantified by means of the optical densities (OD) recorded spectrophotometrically. The test item showed significantly reduced cell viability in comparison to the negative control (mean value: 23 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2. A stepwise procedure is applied to decide on the final classification of the substance, therefore a further in vitro test is necessary.

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:
2018-10-10 to 2018-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: human cornea model
Details on test animals or tissues and environmental conditions:
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.

The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows the identification of a test item's potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that also water insoluble materials may be tested. Prior to use, each plate (6, 12, and 24-well) and its cover will be uniquely identified with a permanent marker by a plate number and/or test article number.
The cytotoxicity of the test article (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test article-treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).
Supplier: MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, Bratislava, Slovakia
Lot No.: 27073
Expiry date: 11 October 2018
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μL (or a sufficient amount to cover uniformly the entire tissue surface)

Duration of treatment / exposure:
The plates with the treated tissue units were incubated for the exposure time of 30 ± 2 minutes at standard culture conditions (37±2°C in an incubator with 5 % CO2, ≥95% humidified atmosphere).
Duration of post- treatment incubation (in vitro):
120 ± 15 minutes
Number of animals or in vitro replicates:
2
Details on study design:

- RhCE tissue construct used:
EpiOcular™ human cell construct (MatTek Corporation), Lot No.: 27073

- Duration and temperature of:
exposure: 30 ± 2 minutes at standard culture conditions (37±2°C in an incubator with 5 % CO2, ≥95% humidified atmosphere)
post-exposure immersion: 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature
post-exposure incubation: 120 ± 15 minutes at standard culture conditions
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm (± 10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752)
- Description of the method used to quantify MTT formazan :
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 μL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±2°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere. Each insert was removed from the 24-well plate after 3 hours ± 15 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) and were stored overnight at 4-10°C in the dark.
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (120 rpm) for approximately 3 hours at room temperature, protect from light. 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. Following the formazan extraction, 200 μL sample(s) from each tube (preferably 2×200 μL if possible) was placed into the wells of a 96-well plate (labelled appropriately) and Absorbance / Optical Density of the samples was determined in a 96-well plate spectrophotometer at the wavelength of 570 nm.
- Description of evaluation criteria used: The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392-492-1722) using the fifteen proficiency chemicals according to OECD Test Guideline No. 492.
- Acceptable variability between tissue replicates for positive and negative controls :
The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
The acceptable percentage viability for positive control (mean of two tissues) is:
- 30 minute exposure: below 50% of control viability
- 6 hours exposure: below 50% of control viability
- Acceptable variability between tissue replicates for the test chemical
The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colourant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: % viability
Run / experiment:
1, mean of two replicates
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

OD values and viability percentages of the controls:

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
Sterile deionized water

1

2.150

92

15

2

2.505

108

mean

2.328

100

 

Positive Control:
Methyl acetate

1

0.370

16

5

2

0.476

20

mean

0.423

18

 

 

OD values and viability percentages of the test item:

Test Item

Optical Density (OD)

Viability (%)

Δ%

Test item

1

2.123

91

3

2

2.188

94

mean

2.155

93

 

standard deviation (SD)

1.97

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation/corrosion potential under the applied testing conditions and is considered as not requiring classification and labelling according to UN GHS (UN GHS No Category).
Executive summary:

A study according to OECD Guideline 492 was conducted to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro. Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item (50 μL/units) and incubated for 30 ± 2 minutes at standard culture conditions (37±2°C in an incubator with 5±1% CO2, ≥95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test items treated tissues were incubated for 120 ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh assay medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±2°C in an incubator with 5±1% CO2 protected from light, ≥95% humidified atmosphere. The formazan precipitated was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated 30 ± 2 minutes. The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 93 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to the eye. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

The skin irritation potential of the test item was tested in vitro on reconstituted human epidermis in the EPISKIN model according to OECD Guideline 439. The test item showed significantly reduced cell viability in comparison to the negative control (1xPBS). Positive (SDS) and negative controls showed the expected cell viability values within acceptable limits. The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2. A stepwise procedure was therefore applied to decide on the final classification of the substance, including a further in vitro test according to OECD Guideline 431, using the EPISKIN model. In this study NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. The results obtained from this in vitro skin corrosion test, indicated that the test item reveals no skin corrosion potential. This result can be used together with the above mentioned results of the in vitro skin irritation study to decide on the final classification of the test item as skin irritating (cat. 2).

Eyes:

A study according to OECD Guideline 492 was conducted to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls respectively. The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 93 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to the eye. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

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 available data, the test item is not considered to be classified and labeled for eye irritation/corrosion, but is considered to be classified for skin irritation into category 2 and labelled with H315 (Causes skin irritation), according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.