Dilithium salicylate

Endpoint summary

Description of key information

Skin Irritation- Irritating to Reconstructed Human Epidermis (OECD TG 439)

Skin Corrosion- Corrosive to Reconstructed Human Epidermis (OECD TG 431)

Eye Irritation- Did not meet criteria for not requiring classification and labelling for eye irritation or serious eye damage in the Reconstructed Human Cornea-like Epithelium Test Method (OECD TG 492)

Eye Corrosion- Meets criteria for chemicals inducing serious eye damage in the Bovine Corneal Opacity and Permeability Test Method (OECD TG 437)

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:

29 Aug 2017 - 31 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: ExxonMobil Research & Engineering (Paulsboro, NJ)
Lot # 17-24168
- Expiration date of the lot/batch: 20 March 2019
-Purity test date: 04 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as supplied

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EpiDerm™ Skin Model closely parallels human skin, and is recommended by test guidelines. Validation studies have reported that tests employing human skin models are able to reliably discriminate between known skin corrosives and non-corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek Corporation (Ashland, MA) EpiDerm™ tissues
- Tissue batch number(s): Lot No. 26787 Kit I
-Shipping Date: 28 Augst 2017
- Delivery date: 29 August 2017
- Date of initiation of testing: 30 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: not specified
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL Dulbecco's Modified Eagle's Medium (DMEM)
- Incubation time: 3 hours
- Spectrophotometer: μQuant Plate Reader (Bio-Tek Instruments, Winooski, VT).
- Wavelength: 540 nM


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was assessed with an MTT QC assay. Application of tissue culture water to the tissues for four hours produced an MTT OD value of 1.7 for Lot No. 26787 Kit I. This value met the criteria of >1.0 but < 3.0 indicating adequate viability of tissues. The historical tissue viability values from the 1996 EpiDerm Database (N=184) for water are mean= 1.47; standard deviation = 0.13; Range ± 2 standar deviatons = 1.21 - 1.73.
- Barrier function: Tissues were exposed to 1% Triton X-100 for 4, 6, 8 and 10 hours. The time of exposure required to reduce the tissue viability (ET-50) using the MTT viability assay is determined (See MatTek EpiDerm MTT ET-50 Protocol) for each lot of tissue. ET-50’s must fall within the range of the 1996 EpiDerm database of 4.77 – 8.72 hours. The ET50 for Lot No. 26787 Kit I was 7.57 hours.
- Morphology: Tissue viability and barrier function tests are wtihin the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers.
- Contamination: The cells used to produced EpiDerm tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants as follows: HIV (oligonucleotide-directed amplification), Hepatitis-B (oligonucleotide-directed amplification), Hepatitis-C (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture)
- Reproducibility: Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness. Tissues were used consistently on the same day, i.e. following overnight storage at 4°C (Wednesday morning) to maintain reproducibility.

NUMBER OF REPLICATE TISSUES: 2

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 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test article was applied to the top of each tissue
- Concentration (if solution): not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 microliters of test article was applied to the top of each tissue
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 microliters of test article was applied to the top of each tissue
- Concentration (if solution): 8N

Duration of treatment / exposure:

Each test article remained in contact with the EpiDerm™ tissue for 3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

Duplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

60 minutes

Value:

13.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

3 minutes

Value:

88.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Viability differences between the two identically treated tissues in all samples and controls at 3 minutes were 0.7% to 5.4%. Viability differences between the two identically treated tissues at 60 minutes were 0.8% to 8.9%. Inter-tissue viability differences at both time points met the acceptance criterion (<30%).

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Skin corrosion is defined as the production of irreversible tissue damage in skin following the application of test material. The mean viability of Dilithium salicylate was 88.2% following a 3 minute exposure and 13.4% following a 60 minute exposure. These findings warrant classification of dilithium salicylate as a Category 1 skin corrosive substance under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

The skin corrosion potential of Dilithium salicylate was assessed MatTek EpiDerm™ SCT tissue model. Tissues were treated in duplicate with the test articles, negative control and positive control for 3 minutes and 60 minutes. Following treatment, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and conversion, and the absorbance of each tissue was measured at 540 nm. The percent viability following a 3 minute and 60 minute exposure was used to determine corrosivity potential. For mean viability ≥50% at 3 minutes and >15%, the test article is determined to be non-corrosive, and ≥50% at 3 minutes but <15% at 60 minutes, the test article is determined to be corrosive. In this case, the mean viability of dilithium salicylate was 88.2% following a 3 minute exposure and 13.4% following a 60 minute exposure. These findings warrant classification of Dilithium salicylate as a Category 1 skin corrosive substance under the  Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Sep 2017 - 23 Oct 2017

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:

28 July 2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Source: ExxonMobil Research and Engineering (Paulsboro, NJ)
Lot #16-41779
Expiration date: 20 March 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received

Test system:

human skin model

Remarks:

MatTek EpiDerm SIT

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTEK EpiDerm 200-SIT
- Tissue batch number(s): 27116 Kit S
- Shipping date: 11 Sep 2017
- Delivery date: 12 Sep 2017
- Date of initiation of testing: 13 Sep 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ~37°C and ~5% CO2
- Temperature of post-treatment incubation (if applicable): ~37°C and ~5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed with PBS and blotted to remove test substance

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: uQuant Plate Reader, Bio-Tek Instruments
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was assessed with an MTT QC assay. Application of tissue culture water to the tissues for four hours produced an MTT OD value of 1.86 for Lot No. 27116 Kit S. This value met the criteria of >1.0 but < 3.0 indicating adequate viability of tissues. The historical tissue viability values from the 1996 EpiDerm Database (N=184) for water are mean= 1.47; standard deviation = 0.13; Range ± 2 standar deviatons = 1.21 - 1.73.
- Barrier function: Tissues were exposed to 1% Triton X-100 for 4, 6, 8 and 10 hours. The time of exposure required to reduce the tissue viability (ET-50) using the MTT viability assay is determined (See MatTek EpiDerm MTT ET-50 Protocol) for each lot of tissue. ET-50’s must fall within the range of the 1996 EpiDerm database of 4.77 – 8.72 hours. The ET50 for Lot No. 27116 Kit S was 7.26 hours.
- Morphology: Tissue viability and barrier function tests are wtihin the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a function stratum corneum, a viable basal cell layer and intermediate spinous and granular layers.
- Contamination: The cells used to produced EpiDerm tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants as follows: HIV (oligonucleotide-directed amplification), Hepatitis-B (oligonucleotide-directed amplification), Hepatitis-C (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture)
- Reproducibility: Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness. Tissues were used consistently on the same day, i.e. following overnight storage at 4°C (Wednesday morning) to maintain reproducibility.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than 50%
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test article was applied to the top of each tissue
- Concentration (if solution): not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliters was applied to the top of the tissue
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliters was applied to the top of the tissue
- Concentration (if solution): 5%

Duration of treatment / exposure:

Tissues were exposed to controls and test substance for approximately 35 ± 1 minutes in a humidified incubator at ~37°C and ~5% CO2, and at room temperature for the remainder of the 60 minute total exposure period.

Duration of post-treatment incubation (if applicable):

Rinsed EpiDerm tissues were returned to the humidified incubator at ~37°C and ~5% CO2 for 42 ± 2 hours.

Number of replicates:

n=3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

24.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The mean OD540 of the negative control tissues was 2.464, and the mean viability of positive control tissues was 2.2%. The standard deviation
(SD) calculated from individual percent tissue viabilities of the three identically-treated replicates was 0.53% for the negative control and 0.39% for
the positive control. All controls passed the acceptance criteria.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In the in-vitro EpiDerm SIT Skin Model, the relative mean tissue viability obtained after a 1-hour treatment with Dilithium salicylate compared to the negative control tissues was 24.5%. These findings warrant classification of Dilithium salicylate as a Category 2 skin irritant substance under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Executive summary:

The MatTek EpiDerm™ SIT skin model was used to assess the potential dermal irritation of dilithium salicylate by determining the viability of the tissues following exposure to the test substance via MTT. Tissues were exposed to test substance and controls for one hour. Pre-testing for coloration and MTT auto-reduction were negative, therefore no data corrections were necessary. The MTT data show the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 2.2% of negative control after 1 hour exposure. Dilithium salicylate reduced tissue viability to 24.5% of negative control after 1 hour exposure. These findings warrant classification of dilithium salicylate as a Category 2 Skin Irritant under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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 August 2017 - 17 August 2017

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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ExxonMobil Research & Engineering (Paulsboro, NJ) Lot # 17-24168
- Expiration date of the lot/batch: 20 March 2019
- Purity test date: 04 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: test substance was soluble in vehicle throughout duration of study

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received
- Final dilution of a dissolved solid, stock liquid or gel: 200 mg/mL of dilithium salicylate in 0.9% sodium chloride

Species:

other: Isolated Bovine Cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The bovine eyes, in a Hanks’ Balance Salt Solution (HBSS) with penicillin-streptomycin, were received from Spear Products on 17 Aug 2017 and transported to MB Research in a refrigerated container.

Vehicle:

physiological saline

Remarks:

0.9% Sodium Chloride Irrigation

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 ml of the 20% (w/v) test article formulation in saline

Duration of treatment / exposure:

Four hours (±10 minutes) at 32 (±1)°C

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

The solution was removed from the anterior chamber and replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco's phosphate buffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator for 90 (±5) minutes.

Number of animals or in vitro replicates:

Three bovine corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of
vascularization, pigmentation, opacity, or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.

QUALITY CHECK OF ISOLATED CORNEAS: A pre-exposure determination of opacity was made for each cornea by measuring each against the blank
supplied by the opacitometer. Any cornea with a value greater than 7 units was discarded.

NUMBER OF REPLICATES: 3 isolated bovine corneas per group

NEGATIVE CONTROL USED: Modified Eagles Medium (MEM)

SOLVENT CONTROL USED (if applicable): 0.9% Saline

POSITIVE CONTROL USED: 20% (w/v) imidazole formulation in 0.9% saline

APPLICATION DOSE AND EXPOSURE TIME: A volume of 0.75 ml of the 20% (w/v) test article formulation in 0.9% saline for four hours (±10 minutes). All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C incubator

TREATMENT METHOD: All corneas were dosed via the closed-chamber method.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Test article formulation, 20% imidazole formulation, MEM or 0.9% saline 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: Differences in light transmission through the control and treated cornea were determined using an OP-KIT opacitometer produced by Electro-Design Corporation of Riom, France. Each treated cornea will be scored in comparison to the blanks provided with the OP-KIT opacitometer .
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD450) Milton Roy/ Spectronic 20-D Colorimeter Model: 333175 Serial No: 33241700
- Others (e.g, pertinent visual observations, histopathology): none

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS Less than or equal to 3 = GHS No Category
IVIS Greater than 3 and less than or equal to 55 = No prediction can be made
IVIS Greater than 55 = Substance causing serious eye damage (GHS Category 1)

Irritation parameter:

cornea opacity score

Run / experiment:

4 hour

Value:

110.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

In vitro irritation score (IVIS) calculated from opacity score

Value:

128.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Corneal surface sloughing was reported in one of three cornea after 4 hour exposure.

The Imidazole positive control IVIS was 91.02, which fell within the acceptance range of 58.10 – 135.86 (± 2 standard deviations of the historical mean).

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The mean corneal opacity, and in-vitro irritation score for Dilithium Salicylate were 110.34 and 128.75, respectively. These findings warrant classification of Dilithium Salicylate as a substance inducing serious eye damage (Category 1 Eye Irritant) under the Regulation (EC) 172/2008 on classification, labelling and packing of substances and mixtures (CLP).

Executive summary:

Dilithium Salicylate was applied to isolated bovine cornea to assess for the potential to induce serious eye damage and irritation. Ocular damage was assessed at 4 hours post-instillation and scored through measurement of corneal opacity and permeability. The mean corneal opacity, and in-vitro irritation score for Dilithium Salicylate were 110.34 and 128.75, respectively. These findings warrant classification of Dilithium Salicylate as a substance inducing serious eye damage (Category 1 Eye Irritant) under the Regulation (EC) 172/2008 on classification, labelling and packing of substances and mixtures (CLP).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2017 - 1 September 2017

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:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ExxonMobil Research & Engineering (Paulsboro, NJ) Lot # 17-24168
- Expiration date of the lot/batch: 20 March 2019
- Purity test date: 04 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable under test conditions
- Solubility and stability of the test substance in the solvent/vehicle: not applicable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received

Species:

human

Details on test animals or tissues and environmental conditions:

RECONSTRUCTED HUMAN CORNEAL-LIKE EPITHELIUM (RHCE) TISSUE
- Model used: MatTEK EpiOcular OCL-200
- Tissue batch number(s): 23495 Kit E
- Shipping date: 28 Aug 2017
- Delivery date: 29 Aug 2017
- Date of initiation of testing: 30 Aug 2017

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: uQuant Plate Reader, Bio-Tek Instruments
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was assessed with an MTT QC assay. Application of tissue culture water to the tissues for four hours produced an MTT OD value of 1.64 for Lot No. 23495 Kit E. This value met the criteria of >1.10 based on the MatTek 1996 QC EpiOcular Database (N=184)indicating adequate viability of tissues.
- Barrier function: Tissues were exposed to 0.3% Triton X-100 for 5, 20, and 60 minutes. The time of exposure required to reduce the tissue viability (ET-50) using the MTT viability assay is determined (See MatTek EpiOcular MTT ET-50 Protocol) for each lot of tissue. ET-50’s must fall within the range of 12.2 – 37.5 hours based on the 1996 QC EpiOcular database (average = 24.9 ± 6.3). The ET50 for Lot No. 23495 Kit E was 7.26 hours.
- Morphology: Tissue viability and barrier function tests are within the acceptable ranges and indicate appropriate formation of the epithelium. Appropriate morphology has also been established for the Validated Reference Method EpiOcular EIT and therefore detailed histological examination does not need to be demonstrated by a test method user for each tissue batch.
- Contamination: The cells used to produced EpiOcular tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants as follows: HIV (oligonucleotide-directed amplification), Hepatitis-B (oligonucleotide-directed amplification), Hepatitis-C (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture)
- Reproducibility: Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness. Tissues were used consistently on the same day, i.e. following overnight storage at 4°C (Wednesday morning) to maintain reproducibility.

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be an eye irritant or substance causing serious eye damage (GHS Category 1 or 2) if the viability following treatment is less than or equal to 60%
- The test substance is considered to be non-irritating (GHS No Category) to eye if the viability following treatment is greater than 60%

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg of the test article were applied to duplicate EpiOcular™ tissues

Duration of treatment / exposure:

The tissues were incubated at 37±1°C, 5±1% CO2 for 6 hours ± 15 minutes.

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

The tissues were rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for 25 minutes. The soaked tissues were then incubated in fresh assay medium at 37±1°C, 5±1% CO2 for 18 hours.

Number of animals or in vitro replicates:

Duplicate EpiOcular™ tissues

Details on study design:

Since the test article was colored, it was assessed for its ability to absorb light at the wavelength used for MTT determination (570 nm). 50 mg of the test article was added to 2 ml of extractant (isopropanol) and incubated for 2 to 3 hours in a six-well plate, at room temperature with shaking. Two aliquots of the testarticle plus extractant from each well were measured at 570 nm using the plate reader. The mean OD570 values of the test article plus extractant was no more than 0.08 after subtraction of blank (undosed isopropanol), so no colorant controls were performed for this test article.

Application/Treatment of test article:
50 mg of the test article were applied to duplicate EpiOcular™ tissues. A negative control (50 μl of TCH2O) and a positive control (50 μl methyl acetate) were each tested concurrently. Each treatment with test article or control was conducted in duplicate. The tissues were then incubated at 37±1°C, 5±1% CO2 for 6 hours ± 15 minutes. After dosing and incubation, the tissues were rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for 25 minutes. The soaked tissues were then incubated in fresh assay medium at 37±1°C, 5±1% CO2 for 18 hours.

Cell Viability Measurement:
At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in Dulbecco's Modified Eagle's Medium). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiOcular™ tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well overnight at room temperature in the dark. Two aliquots of the extracted MTT formazan from each well were measured at 570 nm using a plate reader (μQuant Plate Reader, Bio-Tek Instruments, Winooski, VT).

Irritation parameter:

other: % Viability

Remarks:

mean

Run / experiment:

6 hours

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The R-squared value calculated for the plate reader linearity check was 0.9999, which met the acceptance criterion of greater than 0.999. The mean OD570 of the negative control tissues was 1.734, and the mean relative viability of the positive control tissues was 27.2%. The differences in viability between identically treated tissues were 0.23 - 1.85%. All controls passed the acceptance criteria.

Interpretation of results:

other: GHS Category 1 or 2

Conclusions:

If the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to the established percentage tissue viability cut-off value, no prediction can be made. In this case, further information is required as a positive result in this RhCE test method is not intended to differentiate between GHS Categories 1 and 2.

Executive summary:

The purpose of this study was to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492. Duplicate tissues were treated with DIlithium salicylate for an exposure period of 6 hours, followed by determination of the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of treatment. An irritant is predicted if the mean relative tissue viability of individual tissues exposed to the test substance is ≤ 60% of the mean viability of the negative controls. In this case, the test substance mean relative tissue viability was ≤ 60%, therefore further information is required as a positive result in this RhCE test method is not intended to resolve between Categories 1 and 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Since under REACH, in vitro studies are sufficient for compliance with Annex VII, no key in vivo study has been conducted.

 

EpiDerm SIT (EPI-200), the 3D-model of human epidermal keratinocytes seeded onto a dermal collagen matrix substitute, has been used to evaluate the in vitro irritation of dilithium salicylate. Triplicate human epidermal tissues were treated with dilithium salicylate (25 mg) for a period of 60 minutes and evaluated after 42 hours. The relative mean tissue viability of epidermal keratinocytes exposed to dilithium salicylate was 24.5%, which is below the 50% threshold for skin irritancy. Dilithium salicylate is considered to be an irritant; however further information is required to conclude on the corrosivity potential of dilithium salicylate within the top-down in-vitro testing approach.

Corrosivity has been evaluated in vitro using the human EpiDerm SCT Skin Model. The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. EpiDerm tissues were exposed to dilithium salicylate, skin corrosion was evaluated on the basis of cell viability. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Dilithium salicylate compared to the negative control tissues was 88.2% and 13.4%, respectively. Dilithium salicylate is therefore considered to be corrosive to the skin.

Eye irritation was assessed with EpiOcular EIT (OCL-200), a 3D model of reconstructed human cornea-like epithelium test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Duplicate tissues were treated with dilithium salicylate (50 mg) for a period of 60 minutes and evaluated after 18 hours. The relative mean tissue viability of reconstructed cornea-like epithelium exposed to dilithium salicylate was 3.0%, which is below the threshold of 60% for not requiring classification. Dilithium salicylate is considered to be an eye irritant; however further information is required to conclude on the corrosivity potential of dilithium salicylate within the top-down in-vitro testing approach. The in vitro bovine corneal opacity and permeability (BCOP) test generated an in vitro irritation score (IVIS) of 128.75 for dilithium salicylate. Since the IVIS is > 55, the substance is considered corrosive to the eye.

Justification for classification or non-classification

Dilithium salicylate produced positive evidence of skin corrosion based on results of two in-vitro assays. These findings warrant classification of Dilithium salicylate as a Category 1 Skin Corrosive under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Dilithium salicylate produced evidence of serious eye damage in an in-vitro BCOP assay and warrants classification as Eye Damage Category 1 under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).