[No public or meaningful name is available]

Endpoint summary

Description of key information

In an in vitro skin irritation study according to OECD guideline 439, the test item was found to be non-irritant to skin under the experimental conditions reported. In a in vitro eye irritation study according to OECD guideline 492, the test item was considered non-irritating under the experimental conditions given.

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:

2019-03-21 to 2019-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT)

Version / remarks:

2017-07-11

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Justification for test system used:

The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The EpiDerm™ Skin Irritation Test (SIT) was validated in an international prevalidation study performed by ECVAM and have turned out as a sufficiently promising predictor for skin irritancy potential. Laboratory technical proficiency with the test system according to OECD Test Guideline 439 was demonstrated at Envigo CRS GmbH in March 2014.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number: 28690
- Delivery date: April 02, 2019
- Date of initiation of testing: April 03, 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C and 5 ± 0.5% CO2 (first 35 min) and room temperature (remaining 25 min)
- Temperature of post-treatment incubation: 37 ± 1.5 °C and 5 ± 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: At least 15 times
- Observable damage in the tissue due to washing: Not indicated
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (working solution)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Concurrent negative controls were used in each run to demonstrate that viability of the tissues are within a defined historical acceptance range. Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness can be found in "Attached background material". Furthermore, tissue viability has been checked by the supplier in an MTT QC assay (Acceptance criteria: OD (540-570) [1.0-3.0], Result: OD 2.019 ± 0.08)
- Barrier function: Concurrent positive controls were used in each run to demonstrate that barrier function and resulting tissue sensitivity of the tissues are within a defined historical acceptance range. Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness can be found in "Attached background material". The ET50 should be between 4.0 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria). Furthermore, tissue viability has been checked by the supplier in an ET-50 assay (Acceptance criteria: ET-50 [4.77-8.72 hours], Result: 5.62 hours)
- Contamination: The cells used to produce EpiDerm™ tissues were screened for potential biological contaminants. These included HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and funghi. No contaminations were observed.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues: 2
- Method of calculation used: True viability = Viability of treated tissue – Interference from test chemical = ODtreated viable tissue - ODkilled tissue, where ODkilled tissue = (mean ODtreated killed tissue - mean ODuntreated killed tissue)

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be a skin irritant if the viability after 3 hours exposure is less than or equal 50%.
- The test substance is considered to be non-irritant to skin if the viability after 3 hours exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (undiluted)

NEGATIVE CONTROL
- Amount applied: 30 µL (undiluted)

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %

Duration of treatment / exposure:

60 minutes (35 minutes at 37°C and 25 minutes at room temperature)

Duration of post-treatment incubation (if applicable):

42 hours (incubation medium was changed after 24 hours)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three runs

Value:

80.04

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:

OTHER EFFECTS:
- Visible damage on test system: Not indicated
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed purple colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, see "Attached background material"

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean OD: 1.840)
- Acceptance criteria met for positive control: Yes (mean OD: 0.059)
- Acceptance criteria met for variability between replicate measurements: Yes (SD: 6.2)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This GLP compliant in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test (OECD guideline 439). The test item reduced MTT (pre-test for direct MTT reduction), but it did not change colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed were necessary. The test item, the negative control (DPBS), and the positive control (5% SDS) were applied to triplicate tissue, respectively. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours, the tissues were treated with the MTT solution for 3 hours followed by about 2.5 hours of extraction of the colourant from the cells. The amount of extracted colourant was determined photometrically at 570 nm. After treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.20% thus ensuring the validity of the test system. The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study. Compared to the negative control the mean relative viability was reduced to 80.04% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2019-03-21 to 2019-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals

Version / remarks:

2015-06-29

Deviations:

no

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:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ tissue consists of human-derived epidermal keratinocytes ressembling the human cornea. 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. Laboratory technical proficiency with the test system according to OECD Test Guideline 492 was demonstrated at Envigo CRS GmbH as documented in Envigo CRS project No. 1729900.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. EpiOcular™ kits (Lot No: 27098) and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ∅). A certificate of analysis is available, confirming that cells have been screened for potential biological contaminants (HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria yeast, and other funghi).
- RhCE tissue used, including batch number: All cells used to produce EpiOcular™ are purchased or derived from tissue obtained by MatTek Corporation from accredited institutes. The keratinocyte strain number used was 4F1188 and the Lot No was 27098.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL of the undiluted test item

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

After rinsing, the tissues were immersed in pre-warmed assay medium at room temperature for 12 minutes (post-soak). Afterwards, the tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

Number of animals or in vitro replicates:

2 in vitro replicates

Details on study design:

- Details of the test procedure used: The Reconstructed human Cornea-like Epithelium (RhCE) test method was used. 50 µL of the test item were applied topically on pre- equilibrated EpiOcular™ tissues. The tissues were incubated at 37 ± 1.5 °C and 5 ± 0.5% CO2 (standard culture conditions) for 30 minutes. Afterwards, the tissues were rinsed three times with PBS (approx. 100 mL). After rinsing, the tissues were immersed in 5 mL of Assay Medium for 12 minutes. At the end of the post-soak immersion, the inserts were transferred into the wells of a 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated under standard culture conditions for about 120 minutes. At the end of the post-treatment incubation, tissues were placed into a 24-well plate containing 0.3 mL of MTT solution and incubated for 180 minutes under standard culture conditions. Inserts were removed from the 24-well plate after 180 minutes and then transferred to a 24-well plate containing 2 mL isopropanol in each well. The plates were sealed with parafilm and a standard plate sealer, and were stored at 2-8 °C in the dark for about 16.5 h. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for about 2 hours at room temperature. Then, the tissues were pierced. The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate. The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

- Doses of test chemical and control substances used: 50 µL of undiluted test item and controls

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 minutes under standard culture conditions (exposure), 12 minutes at room temperature (post-exposure immersion) and 120 minutes under standard culture conditions (post-exposure incubation)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Isopropanol and deionized water (colouring test chemicals), deionized water (direct MTT-reduction)

- Number of tissue replicates used per test chemical and controls: Duplicates (test chemical, positive and negative control, freeze-killed tissues), not specified for MTT reduction

- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: The absorbance at 570 nm (OD570) of each well was measured using a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

- Description of the method used to quantify MTT formazan:
Data evaluation included:
1) The mean OD value of the blank control wells (ODblank) for each experiment was calculated.
2) The mean value of the two replicates for each tissue was calculated.
3) The mean ODblank from each mean OD value of the same experiment was subtracted (blank corrected values).
4) The mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test item (TI) was calculated (ODTI, ODNC, ODPC).
5) The mean OD value of the negative control corresponds to 100% viability. Corrected negative control OD = Negative Control OD - ODblank = 100% viability
6) The percent viability of each test group relative to the negative control (= 100%) was calculated: Viability (%) = 100 * (ODTI / ODPC / ODNC) / mean ODNC
7) The difference of the viability values between duplicate tissues was calculated. If the difference is > 20 percentage points (p.p) the test is considered as non-qualified.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category). If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, no prediction can be made from this result in isolation and requires additional information for classification purposes. A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: See “Any other information on materials and methods incl. tables” and “Attached background material”

- Complete supporting information for the specific RhCE tissue construct used: See “Attached background material”

- Reference to historical data of the RhCE tissue construct: See “Attached background material”

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes, see “Attached background material”

- Positive and negative control means and acceptance ranges based on historical data: OD 0.107- 1.174, mean: 0.666 ± 0.276 (positive control) and OD 1.27 – 2.49, mean: 1.94 ± 0.310 (negative control)

- Acceptable variability between tissue replicates for positive and negative controls: See “Any other information on materials and methods incl. tables”

- Acceptable variability between tissue replicates for the test chemical: See “Any other information on materials and methods incl. tables”

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean of run 1 and 2

Value:

80.18

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Mean viability of test item after data correction procedure

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated

DEMONSTRATION OF TECHNICAL PROFICIENCY: See "Attached background material"

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (The OD of the tissues treated with the negative control is > 0.8 and < 2.5 (1.911 and 1.968)).
- Acceptance criteria met for positive control: Yes (The tissue viability of the positive control is below 50% of the negative control viability (21.74%)).

Interpretation of results:

GHS criteria not met

Conclusions:

It can be stated that in this study and under the experimental conditions reported, the test item was considered non-irritating and, thus, does not need to be classified according UN GHS.

Executive summary:

This GLP compliant in vitro study was performed according to OECD guideline 492 to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The test item proved to be an MTT reducer in the MTT pre-test, but did not have intrinsic colour or proved to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. 50 μL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size. Treatment with the positive control induced a decrease in the mean tissue viability compared with the negative control to 21.74%, thus the validity of the test system is ensured. The acceptance criteria were met. The difference of relative viability between the two relating tissues was < 20 p.p. in the same run (for test item tissues, positive and negative control tissues). Irritating effects were not observed following incubation with the test item. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item: 80.18%).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation (RhE), key study, RL1

This GLP compliant in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test (OECD guideline 439). The test item reduced MTT (pre-test for direct MTT reduction), but it did not change colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed were necessary. The test item, the negative control (DPBS), and the positive control (5% SDS) were applied to triplicate tissue, respectively. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours, the tissues were treated with the MTT solution for 3 hours followed by about 2.5 hours of extraction of the colourant from the cells. The amount of extracted colourant was determined photometrically at 570 nm. After treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.20% thus ensuring the validity of the test system. The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study. Compared to the negative control the mean relative viability was reduced to 80.04% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

 

In vitro eye irritation (RhCE), key study, RL1

This GLP compliant in vitro study was performed according to OECD guideline 492 to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The test item proved to be an MTT reducer in the MTT pre-test, but did not have intrinsic colour or proved to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment. Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. 50 μL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size. Treatment with the positive control induced a decrease in the mean tissue viability compared with the negative control to 21.74%, thus the validity of the test system is ensured. The acceptance criteria were met. The difference of relative viability between the two relating tissues was < 20 p.p. in the same run (for test item tissues, positive and negative control tissues). Irritating effects were not observed following incubation with the test item. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the test item: 80.18%).

 

Conclusion: According to the results of an in vitro skin and eye irritation assay, the test item was found to be not irritating to skin and eye.

Justification for classification or non-classification

Based on the available in vitro studies, it is concluded that the test item is not a skin or eye irritant and should not be classified under EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008, as amended for seventeenth time in Regulation (EU) No 2021/849.