[No public or meaningful name is available]

Endpoint summary

Description of key information

ERGP-IEM was tested in an in vitro skin irritation assay and an ex vivo eye irritation assay. The results of the studies were:

ERGP-IEM was not irritating in a skin irritation assay conducted according to OECD 439.

ERGP-IEM was not irritating when tested in an eye irritation assay conducted according to OECD 437.

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:

2020

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:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Company, no information
- Purity, including information on contaminants, isomers, etc.: No information

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Not determined
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not determined
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not determined
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not determined

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Heated to 70C
- Preliminary purification step (if any): No information

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid applied to test system after being heated to 70C

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek EpiDerm Reconstructed Human Epidermis (RhE). Epi-200 RhE Tissues
- Tissue batch number(s): Lot 33026 Kit J
- Production date: No data
- Shipping date: 28 April, 2020
- Delivery date: 28 April, 2020
- Date of initiation of testing: 29 April, 2020

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: At the end of exposure, tissues were washed with a stream of DPBS which completely removed the control solutions but did not completely remove the test substance. Test group tissues maintained viability above the cutoff following the normal timing and steps of the test.
- Observable damage in the tissue due to washing: None.
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL in DMEM
- Incubation time: 3 hours
- Spectrophotometer: Molecular Devices model i3
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : NA

- N. of replicates :
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to the skin if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced to or below 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): 30 uL
- Concentration (if solution): Dosed unchanged

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): Dosed unchanged

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): Dosed unchanged

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

76

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 observed
- Direct-MTT reduction: Not observed
- Colour interference with MTT: Not 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
- Range of historical values if different from the ones specified in the test guideline: Contract research lab maintains records of historical control data for reference.

Interpretation of results:

GHS criteria not met

Conclusions:

ERGP-IEM is identified as a non-irritant material when tested according to OECD 439.

Executive summary:

ERGP-IEM was assessed for its skin irritation potential in a GLP-compliant, OECD Guideline 439 (2019) skin irritation test using the reconstructed human epidermal tissue model, EpiDerm. Tissues were exposed to 30uL of the test substance heated to 70C; the negative control, DPBS; the experimental control, DPBS heated to 70C; and the positive control, 5% Sodium Dodecyl Sulfate, in triplicate for 60 minutes, washed, and given a 42 -hour post exposure incubation. Viability was determined using the MTT assay. Viability is expressed as % of the negative control condition. The controls behaved as expected. Mean tissue viability of ERGP-IEM-exposed tissues was 76% compared to negative controls following a 60 minute exposure. Based on the results of the study, ERGP-IEM is identified as a non-irritant material when tested according to OECD 439.

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:

2020

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: All test articles will be supplied by the Sponsor. Lot# 6252020
- Purity, including information on contaminants, isomers, etc.: No data.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature and humidity and in a dark cabinet.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: No data.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: No data.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No data.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): No data.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test article was used as received, warmed in an incubator at 33ºC prior to dosing.
- Preliminary purification step (if any): No.
- Final concentration of a dissolved solid, stock liquid or gel: Surfactants will be tested at a concentration of 10% weight/volume or volume/volume
(v/v) in 0.9% physiological saline (or other vehicle as specified by the Sponsor). All
other liquids are tested undiluted.

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Cattle. Bovine eyes (at least six months old) will be obtained from an abattoir and will be transported to the laboratory in a refrigerated container containing Hanks’ Balanced
Salt Solution (HBSS) with penicillin-streptomycin.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml
- Concentration (if solution):Solid test articles will be prepared as a 20% solution or suspension in 0.9% physiological saline

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 ml.

Duration of treatment / exposure:

After 10 (±1) minutes, the test article,
ethanol, or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by
washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red.
The anterior and posterior chambers of the holders were then refilled with fresh MEM solution.

Duration of post- treatment incubation (in vitro):

All corneas will be incubated at 32 (±1)°C until immediately prior to the two hour scores, at which time the MEM solution in the anterior and posterior
chambers will be removed and the holders refilled with fresh MEM solution

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : 3

SOLVENT CONTROL USED (if applicable) : 3

POSITIVE CONTROL USED : 3

APPLICATION DOSE AND EXPOSURE TIME : A volume of 0.75 ml of the liquid test article, MEM solution or ethanol will be applied to the epithelium in a manner that ensures the entire cornea will be covered. 10 minute exposure.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 10 (±1) minutes, the test article,
ethanol, or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Criteria defined in TG 437.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

-0.62

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Remarks:

Corrected Mean optical density

Run / experiment:

Mean

Value:

0.003

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

-0.66

Vehicle controls validity:

not examined

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: None.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study, ERGP-IEM is not considered an eye irritant.

Executive summary:

The corneal irritation and damage potential of ERGP-IEM was evaluated in the Bovine Corneal Opacity and Permeability test (BCOP). The study was performed in compliance with EPA GLP 40 CFR 160 and 792, FDA GLP 21 CFR 58, and OECD GLP (1997). The test method was based on OECD 437 (2009). A pretest was performed to measure pre-exposure opacity of the test corneas against the control corneas blanks. The corneas were prepared in cell culture and incubated at 32˚C for at least 1 hour prior to exposure. For the BCOP exposure, corneas (3/test article) were treated with 0.75 mL of undiluted liquid expressed from the test article and then were incubated at 32˚C for 10 minutes. At the end of the exposure, the corneas were washed and incubated in fresh medium for an additional 2 hours. Opacity was evaluated using an opacitometer after the 2 hour post-exposure incubation. Following opacity readings, the cell culture medium was replaced with Na-fluorescein medium and incubated for approximately 90 minutes. Following the 90 minute exposure, permeability was measured. The mean in vitro irritation score (IVIS) was calculated and the scores were classified according to protocol defined categories. Ocular exposure to ERGP-IEM in the test resulted in an IVIS = -0.62, the mean opacity was -0.66 and the mean permeability score was 0.003. Based on the results of the study, ERGP-IEM is not considered an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on the results of the studies, ERGP-IEM is not classified for skin or eye irritation according to GHS.