Reaction mass of sulfonium, dodecylethyl[1-(2-methoxy-2-oxoethyl)-3-oxo-3-(pentyloxy)propyl]-, tetrafluoroborate(1-)(1:1) and sulfonium, dodecylethyl[3-methoxy-1-(2-methoxy-2-oxoethyl)-3-oxopropyl]-, tetrafluoroborate(1-)(1:1) and sulfonium, dodecylethyl[3-oxo-1-[2-oxo-2-(pentyloxy)ethyl]-3-(pentyloxy)propyl]-, tetrafluoroborate(1-)(1:1)

Endpoint summary

Description of key information

In vitro eye irritation and skin irritation studies were conducted on MTDID 47403. The results of the studies were:

 

Eye Irritation: Not irritating when tested according to OECD 437.

 

Skin Irritation: Not corrosive when tested according to OECD 431.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

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: Batch 624730
- Expiration date of the lot/batch: 2017-12
- Purity test date: 05 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Dosed neat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dosed neat

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: No data

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek EpiDerm
- Tissue batch number(s): Lot No. 26717 Kit D and Lot No. 24973 Kit D (Frozen cells)
- Production date:
- Shipping date: No data
- Delivery date: 25 July, 2017
- Date of initiation of testing: 25 July, 2017

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: Each tissue was rinsed with phosphate buffered saline (volume and number of steps were not specified).
- Observable damage in the tissue due to washing: No data

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

NUMBER OF REPLICATE TISSUES: 2 per treatment

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Frozen tissues were run concurently at the 60 minute timepoint for the test article as it reduced MTT. Control frozen tissues were run in parallel.
- Procedure used to prepare the killed tissues (if applicable): No data
- N. of replicates : 2
- Method of calculation used: Corrected Viability = VA – VB - VC
Where:
VA = Mean Viability of live tissues treated with the test article
VB = Mean Apparent Viability of frozen (dead) tissues treated with the test article
VC = Mean Apparent Viability of frozen (dead) tissues treated with TCH2O

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: The average of the values from the duplicate runs were used.

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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 uL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 uL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3, 60 minutes

Duration of post-treatment incubation (if applicable):

3 hour MTT incubation, overnight extractant incubation.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

88.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

77.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results of the study, MTDID 47403 is non-corrosive.

Executive summary:

The in vitro skin irritation/corrosion potential of the test article was evaluated using the MatTek EpiDerm Skin Corrosivity Test. The study was performed in compliance with GLP. The study design was based on the MatTek MTT Effective Time-50 (ET-50) Protocol (protocol no. 711-02) and OECD 431. The test article was dosed neat. Since the test article reduced MTT, frozen (dead) tissues were tested concurrently at 60 minutes and a corrected tissue viablility was calculated. EpiDerm samples were incubated in 6-well tissue culture plates containing Dulbecco's Modified Eagle's Medium (DMEM) for 1 hour. After incubation, 50 uL of the test article was applied to the tissues on top of a mesh spreading aid. The test article remained in contact with the tissue for 3 and 60 minutes. A negative control (50 uL of TCH2O) and a positive control (50 uL of potassium hydroxide solution, 8.0 N) were tested in parallel. Each treatment with test article or control was conducted in duplicate. At the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffer saline (PBS) and transferred to a 24-well microplate containing 300 uL of MTT solution (1 mg/mL MTT in Dulbecco's Modified Eagle's Medium (DMEM); a negative control, 100 uL of TCH2O was tested concurrently. Tissues were then returned to the incubator for a 3-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbency of an aliquot of the extracted MTT formazan was measured in triplicate at 540 nm using a microplate reader. The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate tissues and expressed as percent viability for each sample. A mean viability at 3 minutes of greater than or equal to 50% and a mean viability at 60 minutes greater than or equal to 15% indicates the material was non-corrosive. The positive and negative controls performed as expected which indicated test validity. The corrected cell viability for tissue treated with the test material was 88.9% and 77.6% at 3 and 60 minutes, respectively.  A skin irritation potential cannot be excluded by this test and therefore the substance was classified as a Category 2 skin irritant according to the GHS criteria.

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:

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)

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: Batch 624730
- Expiration date of the lot/batch: 2017-12
- Purity test date: 05 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Dosed neat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dosed neat

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Spear Products
- Number of animals: No data
- Characteristics of donor animals (e.g. age, sex, weight): At least six months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were transported to the laboratory in a solution containing Hanks' Balanced Salt Solution (HBSS) and penicillin-streptomycin in a refrigerated container.
- Time interval prior to initiating testing: No data
- indication of any existing defects or lesions in ocular tissue samples: All 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.
- Indication of any antibiotics used: Penicillin-streptomycin.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

VEHICLE: None

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3 corneas/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.

QUALITY CHECK OF THE ISOLATED 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.

NUMBER OF REPLICATES: 3 per treatment group.

NEGATIVE CONTROL USED: Minimal Essential Media

SOLVENT CONTROL USED: Not applicable

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL

TREATMENT METHOD: Open chamber

POST-INCUBATION PERIOD: Yes, 4 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least two.
- POST-EXPOSURE INCUBATION: 4 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- 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: IVIS Score:
0-3: Non-irritant (Not Classified)
3.1-25: Mild Eye Irritant
25.1 to 55: Moderate Eye Irritant
55.1 and greater: Severe/Corrosive Eye Irritant

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

3.03

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Corrected Mean Optical Density: 0.002
Corrected Mean Opacity Score: 3.00
Mean OD (490 nm) (Permeability): 0.021

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study (IVIS=3.03), MTDID 47403 is considered non-irritating in the Bovine Corneal Opacity and Permeability Test (BCOP).

Executive summary:

The ocular irritation and corrosion potential of MTDID 47403 was evaluated in the Bovine Corneal Opacity Permeability (BCOP) test. The test method was based on OECD 437 and was performed in compliance with OECD GLP. The bovine 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. The dissected corneas were mounted in holders and placed in chambers filled with MEM solution. The entire holder was incubated at 32°C for at least 1 hour but no longer than 2. Corneas (3/group) were treated with 0.75 mL of MTDID 47403 for 10 minutes at 32°C. A positive control (Ethanol) and a negative control (MEM solution), were tested in parallel with the test material. At the end of the exposure period, the corneas were rinsed with a MEM solution containing phenol red; a final rinse was made with MEM without phenol red. Opacity was evaluated using the OP-KIT following the 10 minute exposure. Following opacity readings, the cell culture medium was replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphate buffered saline (PBS) and incubated for approximately 90 minutes at 32°C. Following the 90-minute sodium fluorescein exposure, permeability was measured as the optical density at 490 nm by a spectrophotometer. The In Vitro Irritancy Score (IVIS) for the test material, vehicle control, and positive control was calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density; the IVIS for negative control was performed by adding the mean opacity score to fifteen times the mean optical density. Based on the IVIS score, the test article was classified according to the prediction model described in DB-ALM Protocol No. 127, a modification of the prediction model suggested by Gautheron, et al. (1994). The mean IVIS for the test article was 3.03. The 10 minute mean corrected opacity score was 3.00 and the corrected mean optical density score was 00.002. Controls performed as expected. Based on the results of the study (IVIS=3.03), MTDID 47403 is considered non-irritating in the Bovine Corneal Opacity and Permeability Test (BCOP).

Justification for classification or non-classification

Based on the results of the studies, the test article not classified for eye irritation and is a Category 2 dermal irritant according to GHS.