[No public or meaningful name is available]

Endpoint summary

Description of key information

skin irritation in vitro: not irritating

eye irritation in vitro/ex vivo: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2018

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

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

other: ethyl acetate

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-1001
- Date of initiation of testing: 10 April 2018
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator (37 ± 2 °C) for 20 min- exposure
- Temperature of post-treatment incubation (if applicable): 37 ± 2 °C
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.
PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- A category of UN GHS (Category 2 or Category 1) is predicted if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

The irritating potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied at a 100 % concentration, i.e. 30 µL per insert for 20 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 0.9 % NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 cultures

Value:

ca. 96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

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

Sample No.	Test item	Time [min.]	% Cell Viability
1 -3	negative control NaCl 0.9 %	20	100.00
7 -9	test item	20	96.22
4 -6	positive control	20	3.24

Interpretation of results:

GHS criteria not met

Executive summary:

An in vitro study for predicting non-specific irritating properties of the test item was conducted according to OECD TG 439. The undiluted test item (30 µL per insert) was applied topically to a reconstructed human epidermis model (RhE; epiCS). The cell viability was determined with 96.2 % for the test item as measured by MTT conversion. It is therefore concluded that the test item is not irritating to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

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:

2013

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 °C (± 1 °C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

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 min

Duration of post- treatment incubation (in vitro):

approximately 2 hours

Number of animals or in vitro replicates:

3 corneas per tested material

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 °C (± 1 °C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
yes

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: NaOH (1 %)

APPLICATION DOSE AND EXPOSURE TIME
the liquid test materials were applied pure for 10 min

TREATMENT METHOD:
closed chamber method

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer (BASF OP3.0). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1 mL of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Test item formulations that cause an IVIS value > 55 are classified as seriously damaging the eye (UN GHS Cat 1). Test item formulations that cause IVIS values of ≤ 55 are considered as not seriously damaging the eye (not UN GHS Cat 1).

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

mean

Value:

-0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

tested materials  opacity values per cornea  permeability values per cornea  IVIS per cornea  IVIS mean  negative control (3 corneas) 4.5, 4.2, 4,1  0.010, 0.007, 0.005  4.6, 4.3, 4.2  4.4 (SD 0.2)  positive control (3 corneas) 175.9, 143.7, 188.2  1.679, 1.249, 1.785 201.1, 162.5, 215.0  192.9 (SD 27.2)  test item  1.8, -0.3, -3.7  0.000, 0.003, 0.003  1.8, -0.2, -3.6  -0.7 (SD 2.7)

Interpretation of results:

GHS criteria not met

Executive summary:

The test item was tested in the BCOP test according to OECD TG 437. For determination of corneal damage corneal opacity as well as tissue permeability was measured after a 10 min incubation time. Based on these data, in comparison to the negative control, the In Vitro Irritancy Score (IVIS) value was calculated with -0.7 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

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)

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: three-dimensional human cornea model tissue model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
In principle the EpiOcular™ eye irritation test (EIT) measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). This differentiation would need to be addressed by another tier of a test strategy. As a BCOP has already been conducted and Category 1 can be excluded based on the result, this test can be used to determine, if the substance needs to be classified (Category 2) or not.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium (Part#: OCL-200, Lot No.: 27027; Testing date: 14 Mar 2018).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL per insert

Duration of treatment / exposure:

30 min at standard culture conditions (5 % CO2, 37 °C, 95 % humidity) followed by a post-soak immersion period of about 12 min in fresh medium

Duration of post- treatment incubation (in vitro):

120 min (37 °C, 5 % CO2, 95 % humidity)

Number of animals or in vitro replicates:

each 2 tissues for the test item, positive and negative controls

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Doses of test chemical and control substances used: 50 µg of the neat test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 37 ± 2 °C; 6 h exposure, 25 min. post-soak immersion, 18 h post-treatment incubation

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Killed control and colour control not required.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µL).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %.
- The test chemical is identified as irritant and potentially requiring classification 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 %.

- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

other: cell viability (%)

Run / experiment:

mean

Value:

166.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

15 % viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 439 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation potential of the test item to the eye in vitro.
As the final test item-treated tissue viability was > 60 % (166.6 %) relative to negative control, the test item can be characterized as NOT having eye irritating properties.

Executive summary:

The model used is standardized and commercially available. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The liquid test item was applied topically to the RhCE tissue surface in duplicate for 30 min, followed by an 120 min post-treatment incubation period.

Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is 167 % (rounded).

According to the results of this study the test item was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro study for predicting non-specific irritating properties of the test item was conducted according to OECD TG 439. The undiluted test item (30 µL per insert) was applied topically to a reconstructed human epidermis model (RhE; epiCS). The cell viability was determined with 96.2 % for the test item as measured by MTT conversion. It is therefore concluded that the test item is not irritating to the skin.

The test item was tested in the BCOP test according to OECD TG 437. For determination of corneal damage corneal opacity as well as tissue permeability was measured after a 4 hour incubation time. Based on these data, in comparison to the negative control, the In Vitro Irritancy Score (IVIS) value was calculated with -0.7 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage.

The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The liquid test item was applied topically to the RhCE tissue surface in duplicate for 30 min, followed by an 120 min post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The cell viability was calculated for the test item with 167 % (rounded) relative to the negative control, which was set at 100 %. According to the results of this study the test item was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Justification for classification or non-classification

Based on in vitro studies for skin (OECD 439) and eye irritation (OECD 437 and 492) classification according to EU Regulation 1272/2008 is not required for the registered substance.