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Administrative data

Description of key information

Skin irritation/corrosion:

Two in vitro studies are available.

One in vitro skin corrosion test was performed to evaluate test item ability to induce skin corrosion using a human skin model according to OECD 431. The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions.

Another in vitro skin irritation study was performed using a human three dimensional epidermal model based on the most recent OECD 439. The test item is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

 

Eye irritation:

Bovine Corneal Opacity and Permeability test (BCOP test) was performed based on the most recent OECD guideline 437. The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 5.4 after 10 minutes of treatment.

Another in vitro eye irritation test was performed using Reconstructed Human EpiOcular™Model based on OECD 492. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with test item compared to the negative control tissues was 58%. The test item is potentially irritant or corrosive to the eye.

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:
From 2018-12-03 to 2018-12-07
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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 29642
- Date of initiation of testing: 03 Dec 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubation of 3 minutes at room
temperature, others at 36.4 – 37.1°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were
carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues
(= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Two tissues for a 3-minute and two for a 1-hour exposure, 2 tissues for negative control and 2 tissues for positive control

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 μL

NEGATIVE CONTROL
- Amount(s) applied: 50 μL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration: 8N KOH
Duration of treatment / exposure:
3-minute and 1 hour
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute treatment
Value:
83
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour treatments
Value:
71
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction and Colour interference with MTT: the test item did not interfere with the MTT endpoint because the solutions did not turn blue / purple nor a blue / purple precipitate was observed.

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
Interpretation of results:
other: not corrosive
Conclusions:
In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD 431. The possible corrosive potential of test item was tested through topical application for 3 minutes and 1 hour.

The test item was applied undiluted (50 μL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 4.2% after the 1-hour exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit >= 0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <= 11%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 83% and 71%, respectively. Because the mean relative tissue viability for test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-01-07 to 2019-01-14
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:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model
- Tissue batch number(s): 19 EKIN 001
- Date of initiation of testing: 07 Jan 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive controls.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 ± 0.5 minutes treatment
Value:
32
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction and Colour interference with MTT: The test item did not interact with the MTT endpoint because no color changes and no blue / purple precipitates were observed.

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
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, the test item is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

The objective of this study was to evaluate test item for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)) based on OECD 439. The possible skin irritation potential of test item was tested through topical application for 15 minutes.

The test item was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with test item compared to the negative control tissues was 32%. Since the mean relative tissue viability for test item was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.

All test validation criteria met, the test system functioned properly.

In conclusion, the test item is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
weight of evidence
Study period:
2018-12-11
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)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands),
- Characteristics of donor animals: young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: No
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): 750 μL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Preparation of Corneas: The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- Cornea Selection and Opacity Reading: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.
QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.
NEGATIVE CONTROL USED : physiological saline
POSITIVE CONTROL USED : Ethanol
APPLICATION DOSE AND EXPOSURE TIME
750 μL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: 120 ± 10 minutes at 32 ± 1°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea. The light was measured as illuminance by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Possible pH effects of the test item on the corneas were recorded

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Value:
5.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of 3 treatment
Other effects / acceptance of results:
OTHER EFFECTS:
The corneas treated with test item showed opacity values ranging from 3.4 to 7.5 and permeability values ranging from 0.006 to 0.068. The corneas were translucent (with a clear rim) after the 10 minutes of treatment with test item. No pH effect of the test item was observed on the rinsing medium.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
other: No prediction can be made
Conclusions:
In conclusion, since the test item induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test) based on OECD 437.

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of test item was tested through topical application for 10 minutes.

The test item was applied as it is (750 μL) directly on top of the corneas. All test validation criteria met, it was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 5.4 after 10 minutes of treatment.

In conclusion, since test item induced an IVIS >3 <=55, no prediction on the classification can be made.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-01-14 to 2019-01-18
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:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the
insert in specialized medium.
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): 50 μL
Duration of treatment / exposure:
30 ± 2 minutes
Duration of post- treatment incubation (in vitro):
Post-Soak period: 12 ± 2 minute
Post-incubation period: 120 ± 15 minutes
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used : Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively. Fifty μL of the undiluted test item was added into the 6-well plates on top of the tissues. After the exposure period with test item (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.
- RhCE tissue construct used, including batch number : EpiOcular™, Lot: 27461 Kit A
- Doses of test chemical and control substances used : 50 μL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.
- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2
- Wavelength and band pass used for quantifying MTT formazan : The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Description of the method used to quantify MTT formazan : After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plates containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 mL refrigerated overnight in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.
- Positive and negative control means and acceptance ranges based on historical data : The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5. The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- Acceptable variability between tissue replicates for positive and negative controls : <20%
- Acceptable variability between tissue replicates for the test chemical: <20%
Irritation parameter:
other: tissue viability %
Value:
58
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: potentially irritant or corrosive to the eye
Other effects / acceptance of results:
OTHER EFFECTS:
- direct MTT reduction: Because no color changes were observed it was concluded that test item did not interact with the MTT endpoint.
- color interference: Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0295 and 0.0004, respectively. Therefore it was concluded that the test item did not induce color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
In conclusion, the test item is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or 1).
Executive summary:

The objective of this study was to evaluate the eye hazard potential of test item. For this purpose test item was topically applied on the Reconstructed Human EpiOcularModel based on OECD 492.

The possible eye hazard potential of test item was tested through topical application for 30 minutes. The test item was applied undiluted (50 μL) directly on top of the tissue for 30 ± 2 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

All test validation criteria met, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with test item compared to the negative control tissues was 58%. Since the mean relative tissue viability for test item was below 60% after 30 ± 2 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

In conclusion, test item is potentially irritant or corrosive in the EpiOculartest under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation/corrosion:

The test item is irritant in the in vitro skin irritation test (relative mean tissue viability obtained after 15 ± 0.5 minutes treatment was 32%)

and not corrosive in the in vitro skin corrosion test

According to Regulation (EC) No 1272/2008, section 3.2.2.1, this substance is classified as category 2 for this endpoint.

 

Serious eye damage/eye irritation:

BCOP test: Irritant, IVIS=5.4

In vitro test: potentially irritant or corrosive

According to Regulation (EC) No 1272/2008, section 3.3.2.1, this substance should be classified as Category 2 for this endpoint.