[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2018 - 12 March 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

Deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes

Test system:

isolated skin discs

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

other:

Justification for test system used:

System used to supplant in-vivo testing

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System
EpiDerm™ Reconstructed Human Epidermis Model Kit

Supplier: MatTek
Date received: 22 February 2018
EpiDermTM Tissues (0.63cm2) lot number :25882
Assay Medium lot number:021518TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 uL

Duration of treatment / exposure:

3 min/60 min

Duration of post-treatment incubation (if applicable):

NA

Number of replicates:

2 ea. for postive and negative controls and substance at 3min, 60 min.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ @3 minutes

Value:

ca. 97.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2/@ 60 min

Value:

ca. 111.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

Predicted to be non-corrosive to the skin via employed test system. See attached background material.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. The test item was also found to directly reduce MTT and therefore, an additional procedure using freeze-killed tissues was performed. A third set of controls was included; comprising freeze-killed tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). See attached background material.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2018 - 03 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)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Full validation study of EpiSkinTM recontructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritaion when the end point is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISkin(TM) Reconstituted Human Epidermis Model Kit
- Tissue batch number(s): 18-EKIN-013
- Delivery date: 27 March 2018
- Date of initiation of testing: 28 March 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed using a wash bottle containing PBS with Ca++ and Mg++
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Water killed tissues
- Method of calculation used: No interference due to direct reduction of MTT occurred. The results were therefore not included for quantitative correction.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 15 minutes exposure followed by 42 hour post exposure observation is less than 50%
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure followed by 42 hour post exposure observation is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL of test item added to 2 mL of a 0.3 mg/mL MTT solution

Duration of treatment / exposure:

15-minute exposure period.

Duration of post-treatment incubation (if applicable):

42-hour post-exposure incubation period.

Number of replicates:

Performed in triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test material

Value:

84.7

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: No
- Direct-MTT reduction: test item directly reduced MTT.
- Colour interference with MTT: test item was found to produce a coloured solution which may interfere with the MTT endpoint. Additional colour correction tissues were incorporated, however the results obtained indicated no colour interference occurred.

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

Table 1: Results

Test solution	OD570 of tissues	Mean OD570 of tissues	± SD of OD570	Relative individual viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control	0.522	0.601	0.070	86.9	100	11.6
	0.625			104.0
	0.655			109.0
Positive control	0.039	0.031	0.009	6.5	5.2	1.6
	0.021			3.5
	0.034			5.7
Test Material	0.506	0.509	0.080	84.2	84.7	13.3
	0.591			98.3
	0.431			71.7

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model to measure cytotoxicity following topical exposure to the test item by means of colorimetric MTT reduction assay relative to the negative control (Dulbecco's Phosphate Buffered Saline (DPBS) with Ca⁺⁺ and Mg⁺⁺).

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours.The test item was found to have potential to cause color interference and therefore additional tissues were incorportated for color correction purposes. An assessment of the test item's capability to directly reduce MTT was found to directly reduce MTT and therefore additional non-viable tissues were incorporated intro the testing for correction purposes. A third set of controls was included, compromising freeze-killed tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 -well plate. The optical density was measured at 570 nm.

The relative mean percentage viability of the test item treated tissues was 84.7% after the 15 minute exposure period and 42-hours post-exposure incubation period.

The test item was classified as non-irritant.

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:

key study

Study period:

16 February 2018 - 19 April 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. 6

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The In Vitro irritancy scores are summarized as follows:
Treatment In Vitro Irritancy Score


Test Item 6.0
Negative Control 0.4
Positive Control 43.2

Interpretation of results:

study cannot be used for classification

Conclusions:

The IVIS of 6.0 was determined for the test item.

Executive summary:

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 APR 2018 - 26 NOV 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)

Deviations:

yes

Remarks:

deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes

Irritation parameter:

other: Percent viability

Value:

ca. 46.599

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 46.6% compared to the value of the negative control (classification cut-off: . 60%). The viability resulted from the test item exposed tissues was originally higher than the cut-off value of 60% (68.46 %). The test item was able to reduce MTT by 21.01 percentage points compared to the freeze killed negative control. This value was subtracted from the viability value resulted from the test item exposed tissues in order to correct the MTT interference and therefore the classification changed since 46.599 % is below the classification cut off.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions reported, test item possesses an eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential by means of the Human Cornea Model Test.

Additional tests with viable and freeze-killed tissues were performed, since the test item showed a milky color in the color interference pre-test and proved to be a MTT reducer.

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 relative absorbance compared with the negative control to 42.83%, thus the validity of the test system is ensured.

Irritating effects were observed following 30 minutes incubation. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 46.6% compared to the value of the negative control (classification cut-off: ≤ 60%). The viability resulted from the test item exposed tissues was originally higher than the cut-off value of 60% (68.46 %). The test item was able to reduce MTT by 21.01 percentage points compared to the freeze killed negative control. This value was subtracted from the viability value resulted from the test item exposed tissues in order to correct the MTT interference and therefore the classification changed since 46.599 % is below the classification cut off.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with regulation EC 1272/2008, the test material is classified as H319 Causes serious eye irritation (Category 2). THe substance is not classified for skin irritation or corrosion.