Dilithium glutarate

Endpoint summary

Description of key information

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

 

In-Vitro Skin Corrosion- Not corrosive to reconstructed human epidermis (OECD 431)

In-Vitro Skin Irritation- Not irritating to reconstructed human epidermis (OECD 439)

In-Vitro Eye Corrosion- Not corrosive to isolated bovine cornea (OECD 492)

In-Vitro Eye Irritation- Cytotoxic to reconstructed human cornea-like epithelium (OECD 437); does not meet criteria for chemical not requiring classification for eye irritation/serious eye damage

(Q)SAR- Irritating/Corrosive to eyes >>carboxylic acid and salts

See field "Additional information" for weight of evidence evaluation of eye irritation/serious eye damage potential.

 

 

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:

18 May 2017 - 26 June 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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

White powder
Source: ExxonMobil Research and Engineering (Paulsboro, NJ)
Lot#16-41779
Expiration date: 19 May 2018

Test system:

human skin model

Remarks:

MatTEK EpiDerm SCT

Source species:

human

Cell type:

non-transformed keratinocytes

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:

The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. 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.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTEK EpiDerm 200-SCT
- Tissue batch number(s): 26404
- Delivery date: 23 May 2017
- Date of initiation of testing: 23 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minute exposure at room temperature and 60 minute exposure at ~37°C and ~5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed approximately 20 times with 1 mL DPBS, and gently blotted with cotton swab.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Synergy H4 Spectrophotometer
- Wavelength: 570 nM

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay result OD =1.839 ± 0.079
- Barrier function: ET-50 Asaay Result = 8.04 hrs
- Morphology: Viability and barrier function are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional startum corneum, a viable basal cell layer and intermediate spinous and granular layers.
- Contamination: Biological contaminants not detected

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 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:

~25 mg of the test substance (24.7 mg for 3 minute tissue 1, 25.2 mg for 3 minute tissue 2, 25.3 mg for 3 minute tissue 3, 24.7 mg for 60 minute tissue 1, 24.6 mg for 60 minute tissue 2 and 24.6 mg for 60 minute tissue 3)

Duration of treatment / exposure:

Tissues were exposed to controls and test substance for approximately 3 minutes at room temperature and approximately 60 minutes in a humidified incubator at ~37°C and ~5% CO2.

Number of replicates:

n=3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

87.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

79.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

In the in-vitro EpiDerm SCT Skin Model, the relative mean tissue viability obtained after 3-minute and 1-hour treatments with Dilithium glutarate compared to the negative control tissues was 87.8% and 79.9%, respectively. These findings do not warrant classification of Dilithium Glutarate as a Category 1 Skin Corrosive under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Executive summary:

The MatTek EpiDerm™ SCT skin model was used to assess the potential dermal corrosivity of dilithium glutarate by determining the viability of the tissues following exposure to the test substance via MTT. The objective of this study was to assess the dermal corrosivity of neat (undiluted) Dilithium glutarate. Tissues were exposed to test substance and controls for three minutes and 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, 8N potassium hydroxide (KOH), reduced tissue viability to 29.0% of negative control after 3 minutes exposure and to 9.6% of negative control after 1 hour exposure. The MTT data show that the test substance, dilithium glutarate reduced tissue viability to 87.8% of negative control after 3 minutes exposure and to 79.9% of negative control after 1 hour exposure. These findings do not warrant classification of Dilithium Glutarate as a Category 1 Skin Corrosive 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 - 22 Jan 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:

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: 19 May 2018

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:

The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTEK EpiDerm 200-SIT
- Tissue batch number(s): 27116 Kit S
- 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:

25 mg of test substance

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

Remarks:

mean

Run / experiment:

60 minutes

Value:

97

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:

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:

GHS criteria not met

Conclusions:

In the in-vitro EpiDerm SIT Skin Model, the relative mean tissue viability obtained after a 1-hour treatment with Dilithium glutarate compared to the negative control tissues was 97.0%. These findings do not warrant classification of Dilithium Glutarate as a Category 2 Skin Irritant 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 glutarate by determining the viability of the tissues following exposure to the test substance via MTT. The objective of this study was to assess the dermal irritation of neat (undiluted) dilithium glutarate. 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% sodicum dodecyl sulfate (SDS), reduced tissue viability to 2.2% of negative control after 1 hour exposure. The MTT data show that the test substance, dilithium glutarate reduced tissue viability to 97.0% of negative control after 1 hour exposure. These findings do not warrant classification of dilithium glutarate 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:

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:

16 Aug 2017 - 17 Aug 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:

Dilithium Glutarate CAS 29126-51-0 Lot # 16-41779 in white powder form diluted in 0.9% saline

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:

4 hours (+/- 10 minutes) at 32 (+/-1)°C

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

None

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.
- POST-EXPOSURE INCUBATION:

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 (OD490) 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: The decision criteria as indicated in the TG was used

Irritation parameter:

cornea opacity score

Value:

2.67

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

In vitro irritation score (IVIS) calculated from opacity score

Value:

2.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corneal opacity, and in-vitro irritation score for Dilithium glutarate were 2.67 and 2.75 respectively. These findings do not warrant classification of Dilithium glutarate as a substance inducing serious eye damage (Category 1 Eye Irritant) under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

Dilithium glutarate (neat) 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 glutarate were 2.67 and 2.75 respectively. These findings do not warrant classification of Dilithium glutarate as a Category 1 eye irritant under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Dilithium Glutarate
CAS 29126-51-0
Lot # 16-41779
Expiration Date: 19 May 2018
Description: White Powder

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

Duration of treatment / exposure:

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

Observation period (in vivo):

Not Applicable

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:

2 replicate 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 test article 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: Mean Tissue Viability (%)

Value:

4.5

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 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%.

Interpretation of results:

other: 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 testing with other test methods will be required because a positive result for this RhCE test method cannot resolve between 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 the test item 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 testing with other test methods will be required because a positive result for this RhCE test method cannot resolve between Categories 1 and 2.

Reference 3

Endpoint:

eye irritation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES v.2.27.17

2. MODEL (incl. version number)
Eye irritation v.04 June 2015

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
[Li]{+}.O{-}C(=O)CCCC(=O)O{-}.[Li]{+}

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version: OASIS TIMES v.2.27.17
- Model(s) used: Eye irritation v.04 June 2015
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

Specific details on test material used for the study:

SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
[Li]{+}.O{-}C(=O)CCCC(=O)O{-}.[Li]{+}

Irritation parameter:

other: QSAR Prediction

Remarks on result:

other: Irritating/Corrosive to eye >> Carboxylic acids and salts

Interpretation of results:

study cannot be used for classification

Conclusions:

Dilithium glutarate is predicted to be an eye corrosive/irritant based on the presence of a carboxylic acid structural fragment. There is no available classification criteria for QSARs and therefore this study should only be used as supporting information within a weight of evidence.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

A weight of evidence approach was applied to determine the classification for eye irritation potential of dilithium glutarate using the framework for skin irritation/corrosion presented in OECD guidance document No. 203.

Module 1. Existing human data:

Not available

 

Module 2. In-vivo study:

Not available

 

Module 3. In-vitro corrosion data:

Due to the presence of a carboxylic acid functional group, dilithium glutarate was tested for eye irritation potential in a "top-down" approach. The eye corrosion potential of dilithium glutarate was evaluated using the in vitro bovine corneal opacity and permeability (BCOP) test according to OECD 437 which generated an in vitro irritation score (IVIS) of 2.75. Since the IVIS score was <3, Dilithium Glutarate is not considered corrosive to the eye. The study was given a data quality Klimisch score of 1, and is considered relevant and capable of identifying substances inducing serious eye damage (Category 1 irritant). This data is consistent with other in-vitro data suggesting that dilithium glutarate is not corrosive to the skin (module 5). The in-vitro corrosion data suggest that the substance is not corrosive to the eye and has a high weight in the final evaluation.

 

Module 4. In-vitro irritation data:

Eye irritation was assessed with the EpiOcular EIT (OCL-200) according to OECD TG 492, a test method based on a 3D model consisting of reconstructed human cornea-like epithelium and used for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. The relative mean tissue viability of reconstructed cornea-like epithelium exposed to dilithium glutarate was 4.5%, which is below the threshold of 60% for not requiring classification. The study was given a data quality Klimisch score of 1, and is considered relevant and capable of identifying substances not requiring classification as a substance causing serious eye damage or irritation (UN GHS no category). This data is consistent with the QSAR prediction (module 7) of Dilithium Glutarate as an eye irritant and has a high weight in the final evaluation.

 

Module 5. Other in-vivo and in-vitro data

Dilithium Glutarate was evaluated for skin irritation/corrosion in two in-vitro assays using reconstructed human epidermis (OECD TG 431 and 437). Both studies indicated that dilithium glutarate was not corrosive or irritating to the skin. Both studies were assigned a Klimisch quality score of 1 and considered capable of identifying substance that should be classified as Category 1 or 2 skin irritants. While the data does not directly address irritation/corrosion of the eye, information obtained from skin irritation (Category 2) may be considered to support the classification for eye irritation (Category 2) per the precautionary principle. Due to the limited coverage of relevant parameters especially in the situation of negative dermal irritation/corrosion these studies are given lower weight in the final assessment.

 

Module 6. Physico-chemical Properties:

Test data on the pH of dilithium glutarate is not available, but as a weak acid is not expected to fall into the extremes (pH <2 or >11).

 

Module 7. Non-testing methods ((Q)SAR, grouping, bridging & additivity approaches):

A (Q)SAR prediction for eye irritation/corrosion was generated using the Eye Irritation v.04 June 2015 model of the OASIS TIMES v.2.27.17 software. Dilithium glutarate is predicted to be an eye corrosive/irritant based on the presence of a carboxylic acid structural fragment. The QSAR prediction is given a Klimisch score of 2 (reliable with restrictions) as the results are derived from a valid (Q)SAR model with adequate and reliable documentation/justification but not (completely) falling into the applicability domain. The (Q)SAR model is considered capable of predicting irritation to the eye or organic substances. This data is consistent with the in-vitro irritation data. The (Q)SAR prediction suggests eye irritation or corrosion but does not allow discrimination and due to the chemical falling outside of the structural applicability domain, it is given a lower weight in the final evaluation.

 

Overall Conclusion:

OECD Guidance Document No. 203 recommends that in a weight of evidence approach where there is inconsistent data of differing weights, "a weight of evidence conclusion may be drawn according to the evidence carrying the highest weight". Although the physical-chemical properties and information on dermal irritation/corrosion suggest that Dilithium glutarate is not irritating, these pieces of information are given a lower weight in the overall assessment than the in-vitro eye irritation test data. Further, while EChA guidance on application of CLP (R.7.2.9.1) indicates that OECD TG 492 is “not recommended for the identification of eye irritants (Category 2)”, Dilithium glutarate induced a clear cytotoxic response that was well below the cutoff criteria and based on available information does not rule out the potential for mild eye irritation in-vivo. Since no new in-vivo testing is considered at Annex VII, this data is considered to support the classification for eye irritation (Category 2) as a precautionary principle.

Justification for classification or non-classification

Dilithium glutarate did not produce any evidence of skin irritation or corrosion in two in-vitro assays. These findings do not warrant classification of Dilithium Glutarate as a Category 1 Skin Corrosive or Category 2 Skin Irritant under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Dilithium glutarate did not produce evidence of serious eye damage in an in-vitro BCOP assay and does not warrant classification as Eye Damage Category 1 under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP). A weight of evidence evaluation was conducted to determine if Category 2 Eye Irritant classification was warranted. Based on the weight of evidence, the available data is considered to support the classification for eye irritation (Category 2) under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP)