Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)

Endpoint summary

Description of key information

Skin Irritation/Corrosion: Not irritant to skin ; OECD 439: F.E. Lacey., 2019

Skin Irritation/Corrosion: Not corrosive to skin ; OECD 431:F.E. Lacey., 2019

Eye Irritation/Corrosion: no categorisation for irritation could be made; OECD 437: R. Brown., 2019

Eye Irritation/Corrosion: no prediction can be made on corrosive or mildly irritating to the eye; OECD 492: A. Sokolowski., 2019

Eye Irritation/Corrosion: Not irritant or corrosive to the eye (QSAR - 2019; K.B. Paul)

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:

06 December 2018 - 08 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDermTM Tissues (0.63cm2 lot number 28672)

Cell source:

other: Matket model kit (112918MSD)

Justification for test system used:

Guideline specific test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue batch number(s): 28672
- Production date: not reported
- Shipping date: not reported
- Delivery date: 04 December 2018
- Date of initiation of testing: not reported

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

REMOVAL OF TEST MATERIAL AND CONTROLS -Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Labtech LT-4500 microplate reader and LT-com analysis software.
- Wavelength: 570nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: A comparison of laboratory historical data for negative and positive controls was made to verify the functioning of the test system.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: Coefficient of Variation between tissue replicates was ≤ 30%.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not reduce MTT.

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):25mg
- Concentration (if solution): unchanged - applied as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): n/a

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

Duration of treatment / exposure:

3 and 60 mins

Duration of post-treatment incubation (if applicable):

3 h MTT incubation followed by overnight isopropanol extraction.

Number of replicates:

2 per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

103.6

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 minute

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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): unchanged- applied as supplied.

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25mg
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 mg
- Concentration (if solution): 5 % w/v


- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
The mean OD570 for the negative control treated tissues was 1.963 for the 3-Minute exposure period and 1.996 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 2.9% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Table 1       Relative mean viabilities for each treatment group.

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minutes	100*	4.2	103.6
60 minutes	100*	2.9	97.0

*The mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin under the conditions of the test.

Executive summary:

OECD 431 (2019) - The skin corrosivity potential of the test item, CPI-110A was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD Guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a Labtech LT-4500 microplate reader and LT-com analysis software.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 103.6 and 97.0 %, respectively. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2018 - 17 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Episkin RHE model supplied by SkinEthic Laboratories, France.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 18-EKIN-050
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 11 December 2018
- Date of initiation of testing: 17 December 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a, test item did not reduce MTT.
- Procedure used to prepare the killed tissues (if applicable): n/a
- N. of replicates : n/a
- Method of calculation used: n/a

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is equal to or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 min test item exposure

Duration of post-treatment incubation (if applicable):

42 h followed by 3 h MTT incubation

Number of replicates:

3 per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 mins exposure

Value:

105.9

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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg
- Concentration (if solution): unchanged- applied as supplied.

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 mg
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 mg
- Concentration (if solution): 5 % w/v


- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
The relative mean tissue viability for the positive control treated tissues was 4.0% relative to the negative control treated tissues and the standard deviation value of the viability was 0.5%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.860 and the standard deviation value of the viability was 7.1%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.5%. The test item acceptance criterion was therefore satisfied.

Table 1. Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item.

Item	OD570of tissues	Mean OD570of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.929	0.860	0.062	108.0	100*	7.1
	0.810			94.2
	0.842			97.9
Positive Control Item	0.036	0.034	0.004	4.2	4.0	0.5
	0.029			3.4
	0.036			4.2
Test Item	0.951	0.911	0.064	110.6	105.9	7.5
	0.944			109.8
	0.837			97.3

OD =Optical Density


SD =Standard deviation
  

*= The mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test item was classified as non-irritant (UN GHS classification not met).

Executive summary:

OECD 439 (2019) - The skin irritation potential of the test item, CPI-110A was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

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

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

Mean viability of tissues exposed to the test substance after 15 minutes were 105.9 %. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is not considered to be irritant to the skin.

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:

10 January 2019 - 10 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed under GLP and all relevant validity criteria were met

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:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Corneas from bovine eyes were obtained from a local abattoir.
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, completely immersed in Hanks Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin at 100 IU/mL and streptomycin at 100 µg/mL added to Hanks Balanced Salt Solution during transportation.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Vehicle:

unchanged (no vehicle)

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): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

240 minutes.

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

90 minutes.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


QUALITY CHECK OF THE ISOLATED CORNEAS

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

NUMBER OF REPLICATES

3 per treatment group

NEGATIVE CONTROL USED

The negative control substance was 0.9% sodium chloride solution (Batch; 18/10BB1B, exp: 10 April 2019) supplied by Baxter.

SOLVENT CONTROL USED (if applicable)

N/A

POSITIVE CONTROL USED

The positive control substance was Imidazole >99% (Batch; 162411, exp: 01 August 2021) Supplied by Fisher Scientific.

APPLICATION DOSE AND EXPOSURE TIME

0.75 mL applied to each cornea and incubated at 32 ± 1 °C for 240 minutes.

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION: 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Labtech LT-4500 microplate reader
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD492)
- Others (e.g, pertinent visual observations, histopathology): (please specify) The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.

Irritation parameter:

in vitro irritation score

Run / experiment:

3 runs

Value:

1.2

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: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: n/a

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements & IVIS Score

Treatment	Cornea No.	Opacity				Permeability		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	5	5	0		0.003
	2	4	5	1		0.005
	3	5	6	1		0.004
				0.7*		0.004¨		0.7
Positive Control	6	5	75	70	69.3	1.264	1.260
	7	5	79	74	73.3	1.490	1.486
	8	3	68	65	64.3	1.810	1.806
					69.0!		1.517!	91.8
Test Item	10	4	4	0	0.0	0.054	0.050
	11	3	6	3	2.3	0.015	0.011
	12	6	7	1	0.3	0.003	0.000
					0.9·		0.020·	1.2

OD = Optical density

* = Mean of the post-treatment & pre-treatment values

" = Mean permeability

! = Mean corrected value

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that under the condition of this study, the test substance produced an IVIS score of 1.2 and therefore no categorisation for irritation could be made and the substance does not meet the criteria for classification in accordance with UN GHS or EU CLP.

Executive summary:

OECD 437 (2019) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using CPI-110A in accordance with 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" (2017).

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for


240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability), were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 492 nanometers (OD492). The mean corrected opacity reading and permeability readings for the test item were 0.9 and 0.020, resulting in an In Vitro Irritation Score (IVIS) of 1.2

It was concluded that under the condition of this study, the test substance produced an IVIS score of 1.2 and therefore no categorisation for irritation could be made and the substance does not meet the criteria for classification in accordance with  UN GHS or EU CLP.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 February 2019 - 21 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed under GLP and all relevant validity criteria were met

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcularTM kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcularTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm2) are cultured on specially prepared cell culture inserts (MILLICELL , 10 mm ø ).
EpiOcularTM tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24- well plate on Tuesday. On day of receipt of the EpiOcularTM tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6- well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcularTM tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (about 17 hours).

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): 50 mg
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

Each group tested in duplicate

Details on study design:

Details of the test procedure used

Pre-Test Assessments
For this purpose, approx. 50 mg of the test item were added to 2.0 mL of isopropanol and shaken for 3 hours at room temperature. 2.0 mL of isopropanol were used as control. Additionally, approx. 50 mg of the test item were added to 1.0 mL of deionised water and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour. 1 mL of deionised water was used as control. Since the test item was not completely soluble in isopropanol it was centrifuged, and 200 μL aliquots of the supernatant were used for measurement.
According to guideline the absorbance of all samples (200 μL aliquots) were measured in duplicates at 570 nm (OD570) with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
Since the OD of the test item at 570 nm, after subtraction of the OD for isopropanol or water was < 0.08 (which corresponds to approximately 5% of the mean OD of the negative control) the test item was considered as not interacting with the MTT measurement and an additional test on additional viable tissues (without MTT addition) were not necessary.
Approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for 3 hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently.
Since the MTT solution colour did not turn blue/purple, the test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

Main Test

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.

After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.

At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 25 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.

At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 18 hours the MTT assay was performed.

At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2.0 mL of isopropanol in each designated well so that no isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer and were immediately extracted and shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.

The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 27092). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 mg applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of the method used to quantify MTT formazan: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model.
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category).
If the test item- sue viability, no prediction can be made from this result in isolation and requires additional information for classification purposes.
According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: % tissue viability based on spectrophotometric mean relative absorbance of MTT solution at 570 nm

Run / experiment:

Mean value

Value:

13.37

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.810 and 2.006)
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (6.73%)
- Range of historical values if different from the ones specified in the test guideline: n/a
-The difference of viability between the two relating tissues of a single item is < 20% (values between 0.79 p.p and 10.51 p.p) in the same run (for positive and negative control tissues and tissues of single test items)

Table 1.Assessment of Colour Interference with the MTT endpoint

Treatment Group	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Evaluation Mean OD570(blank corrected) > 0.08
Blank Aqua Deion.	0.036	0.036	0.036
Test Item + Aqua Deion.	0.050	0.072	0.061	0.025	No
Blank Isopropanol	0.036	0.035	0.036
Test Item+ Isopropano	0.037	0.037	0.037	0.001	No

Table 2. Results after treatment for 6 hours with CPI-110A and the controls.

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean [OD570] of T1 and T2	Tissue viabil.* [%]	rel. viabil. of T1 and T2**	Diff. of viabil. between T1 and T2 [p.p.]
Blank Aqua Deion.		0.035	0.035	0.035
Negative Control	1	1.837	1.853	1.845	1.810	1.908	100.00	94.9	10.29
	2	2.064	2.019	2.042	2.006			105.1
Positive Control	1	0.158	0.155	0.156	0.121	0.128	6.73	6.3	0.79
	2	0.173	0.169	0.171	0.136			7.1
Test Item	1	0.193	0.187	0.190	0.155	0.255	13.37	18.6	10.51
	2	0.394	0.387	0.391	0.355			8.1

* mean of 2 replicates after blank correction

** relative absorbance = ((100 • test item absorbance) / mean absorbance of the negative control)

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for CPI-110A.

Executive summary:

OECD492 (2019) - in vitro study was performed to assess the eye irritation potential of CPI-110A by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 μL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with CPI-110A. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (determined value for the test item 13.37%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for CPI-110A.

Reference 3

Endpoint:

eye irritation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

26 July 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

Most recent version of OECD Toolbox and Toxtree were used

Justification for type of information:

1. SOFTWARE

Toxtree 3.1.0 and OECD QSAR Toolbox 4.3

2. MODEL (incl. version number)

BfR decision support system (BfR DSS. BCFBAF v3.01; November 2012


3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
:
Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-): F[Sb](F)(F)(F)(F)(F)S(c1ccccc1)(c1ccccc1)c1ccc(Sc2ccccc)cc1
Hexafluoroantimonate:F[Sb](F)(F)(F)(F)(F)
Sulfonium diphenyl[4-(phenylthiol)phenyl]: S(c1ccccc1)(c1ccccc1)c1ccc(Sc2ccccc)cc1


4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL

Please refer to attached justification.



5. APPLICABILITY DOMAIN

Please refer to attached justification.



6. ADEQUACY OF THE RESULT

Please refer to attached justification.

Qualifier:

no guideline required

Principles of method if other than guideline:

The determination of eye irritation/corrosion potential of diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-) was performed by the Toxtree 3.1.0 and OECD QSAR Toolbox 4.3. The BfR rule-based decision support system classifies a compound as having the potential to cause adverse effects to the eye. All modelling was based on the input of SMILES notation for each constituent using SMILES descriptors. Descriptor values were assigned from the experimental information, which were for the purposes of the fulfilment of REACH (Regulation (EC) No. 1907, 2006), physical/chemical data requirements and from the structure of the chemical(s) itself. When not available the values were predicted using OECD QSAR toolbox v4.3.

GLP compliance:

no

Remarks:

no guideline required

Specific details on test material used for the study:

Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-): F[Sb](F)(F)(F)(F)(F)S(c1ccccc1)(c1ccccc1)c1ccc(Sc2ccccc)cc1

Hexafluoroantimonate = F[Sb](F)(F)(F)(F)(F)

Sulfonium diphenyl[4-(phenylthiol)phenyl] = S(c1ccccc1)(c1ccccc1)c1ccc(Sc2ccccc)cc1

Species:

other: N/A - in silico assessment

Strain:

other: N/A - in silico assessment

Details on test animals or tissues and environmental conditions:

N/A - in silico assessment

Amount / concentration applied:

N/A - in silico assessment

Duration of treatment / exposure:

N/A - in silico assessment

Observation period (in vivo):

N/A - in silico assessment

Duration of post- treatment incubation (in vitro):

N/A - in silico assessment

Number of animals or in vitro replicates:

N/A - in silico assessment

Details on study design:

N/A - in silico assessment

Irritation parameter:

other:

Run / experiment:

QSAR run 1 result

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: QSAR reslut is qualittive not quantitative

Remarks:

No value assignable - result is unknown/ not classified - see discussion

Other effects / acceptance of results:

N/A - in silico assessment

Irritant / corrosive response data:

N/A - in silico assessment

Other effects:

N/A - in silico assessment

Table 2.0: Constituent names, Inputs (SMILES) and predictor values

Constituent Number	Constituent Name (IUPAC)	SMILES Notation	Molecular Weight	Melting Point	Log KOW^§	Water solubility	Lipid solubility	Irritating or corrosive to the eye^§
1	Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)	F[Sb](F)(F)(F)(F)(F)S(c1ccccc1)(c1ccccc1)c1ccc(Sc2ccccc)cc1	607	122°C	8.3* (OECD QSAR Toolbox 4.3) And > 9 (Toxtree v3.1.0)	1.18 x 10^-5g/L*	-	Not R41 (OECD QSAR tollbox 4.3) Not R34, R35 R36 or R41 (Toxtree 3.1.0) Unknown
2	Sulfonium diphenyl[4-(phenylthiol)phenyl]	S(c1ccccc1)(c1ccccc1)c1ccc(Sc2ccccc)cc1	373	196°C*	-0.43	< 8.45 x 10^-4g/L	3.17 x 10^-2	Unknown
3	Hexafluoroantimonate	F[Sb](F)(F)(F)(F)(F)	236	-131°C*	0.71*	< 1.02 x 10^-4g/L	-	Unknown

*Predicted value

^§Some discrepancies in the predicted output for logK_OWexist between the OECD toolbox v4.3 and Toxtree v3.1.0. Both leading to different conclusions for exclsion, but, neither leading to classification.

Red text= value driving prediction

Substance 1 (associated form) fell within the applicability domain of the physical chemical rulebase as the log KOW > 9 C (although predictions via OECD QSAR toolbox v4.3 are lower, at 8.3). Using the OECD QSAR toolbox v.4.3 predictions, the substance falls within the applicability domain of the physical chemical rulebase for water solubility < 0.00002 g/L.

The latter predicts no eye damage but does not exclude irritation, the former predicts no skin corrosion, eye damage or irritation. The substance fell outside the applicability domain of the structural rulebase (i.e. it was not possible to include irritating or corrosive effects based on the current chemical space included in the model).

Substance 2 (dissociated cation) fell outside of the applicability domain of the physical chemical rulebase (i.e. it was not possible to exclude irritating or corrosive effects), it also fell outside the applicability domain of the structural rulebase (i.e. it was not possible to include irritating or corrosive effects based on the current chemical space included in the model).

Substance 3 (dissociated anion) fell outside of the applicability domain of the physical chemical rulebase (i.e. it was not possible to exclude irritating or corrosive effects), it also fell outside the applicability domain of the structural rulebase (i.e. it was not possible to include irritating or corrosive effects based on the current chemical space included in the model).

Note that falling outside of the applicability domain in this model does not mean the model is entirely inappropriate. For the physical/chemical rulebase falling outside of the domain simply means it is not possible to conclusively prove that the chemical is not classified for eye irritation or damage. For the structural rulebase, falling outside the domain simply means it is not possible to conclusively prove that the chemical is classified for eye irritation or damage. Not falling in the applicability domain for either, therefore, results in 3 possibilities:

 

1.    The substance is not classified but physical chemical rules are not applicable due to other mitigating factors

2.    The substance is not classified and, therefore, the structure is not captured by the database

3.    The substance is classified but the structural cause has not yet been documented

 

Based on the large number of chemical structures (> 1000) used in the training set, it is less likely that bullet 3 is true.

The results should be considered in relation to corresponding information presented and in accordance with the tonnage driven information requirements of REACH Regulation (EC) 1907/2006 in a weight of evidence. Specifically:

i. The substance was not considered to be irritating or corrosive to the eye in a Bovine Corneal Opacity and Permeability (BCOP) Assay (OECD, 437);

ii. No conclusion on eye irritation or corrosion could be made during an In vitro Human Cornea Model Test (OECD, 492);

iii. The water solubility is considered to be:

1.18 x 10-5 g/L for substance 1 (OECD QSAR toolbox v4.3)

8.45 x 10-4 g/L for substance 2 (OECD 105)

1.02 x 10-4 g/L for substance 3 (OECD 105)

iv. The melting point is considered to be:

122 degrees C for substance 1 (OECD 10)

196 degrees C for substance 2 (OECD QSAR toolbox v4.3)

-131 degrees C for substance 3 (OECD QSAR toolbox v4.3);

v. The log Kow is considered to be: 8.3 (OECD QSAR toolbox v4.3) or > 9 (Toxtree v3.1.0) for substance 1 -0.43 for substance 2 (OECD 107) 0.71 for substance 3 (OECD QSAR toolbox v4.3);

vi. An absence of any exclusion or inclusion criteria, taken in combination with the weight of evidence from experimental data, is likely indicative of the chemical having no damaging or irritating effects to the eye; 

vii. Antimony although not within the chemical applicability domain of the model, is not classified as an eye irritant or damager on the C&L database (https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/82755), therefore, any irritating effect would be caused by other atoms or moieties if alerted;

viii.  The weight of evidence indicates the substance should not be classified for eye irritation or corrosion under Regulation (EC) 1272/2008 as amended.

Interpretation of results:

other: (QSAR) derived endpoint

Conclusions:

There was no prediction for eye irritation or eye damage, further some exclusion rules were met during the assessment, therefore the chemical is considered non-irritant or damaging.

The prediction is adequate for the Classification and Labelling and/or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006 in a weight of evidence approach and to aid in the fulfilment of the data requirements of Regulation (EC) No. 1907/2006: Annex VII.

Executive summary:

The determination of eye irritation/corrosion potential of Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium  hexafluoroantimonate(1-) was performed by the Toxtree 3.1.0 and OECD QSAR Toolbox 4.3. The BfR rule-based decision support system classifies a compound as having the potential to cause adverse effects to the eye. All modelling was based on the input of SMILES notation for each constituent using SMILES descriptors. Descriptor values were assigned from the experimental information, which were for the purposes of the fulfilment of REACH (Regulation (EC) No. 1907, 2006), physical/chemical data requirements and from the structure of the chemical(s) itself.   When not available the values were predicted using OECD QSAR toolbox v4.3.

The assessment indicates that the prediction is suitable for the regulatory conclusion set out in section 4.1. This conclusion is with due regard to REACH Annex VI-XI.

The prediction is adequate for the Classification and Labelling and/or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006 in a weight of evidence approach and to aid in the fulfilment of the data requirements of Regulation (EC) No. 1907/2006: Annex VII.

The results should be considered in relation to corresponding information presented and in accordance with the tonnage driven information requirements of REACH Regulation (EC) 1907/2006 in a weight of evidence. Specifically:

i. The substance was not considered to be irritating or corrosive to the eye in a Bovine Corneal Opacity and Permeability (BCOP) Assay (OECD, 437);

ii. No conclusion on eye irritation or corrosion could be made during an In vitro Human Cornea Model Test (OECD, 492);

iii. The water solubility is considered to be:

1.18 x 10-5 g/L for substance 1 (OECD QSAR toolbox v4.3)

8.45 x 10-4 g/L for substance 2 (OECD 105)

1.02 x 10-4 g/L for substance 3 (OECD 105)

iv. The melting point is considered to be:

122 degrees C for substance 1 (OECD 10)

196 degrees C for substance 2 (OECD QSAR toolbox v4.3)

-131 degrees C for substance 3 (OECD QSAR toolbox v4.3);

v. The log Kow is considered to be: 8.3 (OECD QSAR toolbox v4.3) or > 9 (Toxtree v3.1.0) for substance 1 -0.43 for substance 2 (OECD 107) 0.71 for substance 3 (OECD QSAR toolbox v4.3);

vi. An absence of any exclusion or inclusion criteria, taken in combination with the weight of evidence from experimental data, is likely indicative of the chemical having no damaging or irritating effects to the eye; 

vii. Antimony although not within the chemical applicability domain of the model, is not classified as an eye irritant or damager on the C&L database (https://echa.europa.eu/information-on-chemicals/cl-inventory-database/-/discli/details/82755), therefore, any irritating effect would be caused by other atoms or moieties if alerted;

viii.  The weight of evidence indicates the substance should not be classified for eye irritation or corrosion under Regulation (EC) 1272/2008 as amended.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion/irritation

OECD 439 (2019) - The skin irritation potential of the test item, CPI-110A was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

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

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

OECD 431 (2019) - The skin corrosivity potential of the test item, CPI-110A was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD Guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a Labtech LT-4500 microplate reader and LT-com analysis software.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 103.6 and 97.0 %, respectively. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

In vitro Eye corrosion/irritation

OECD 437 (2019) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using CPI-110A in accordance with 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" (2017).

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for


240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability), were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 492 nanometers (OD492). The mean corrected opacity reading and permeability readings for the test item were 0.9 and 0.020, resulting in an In Vitro Irritation Score (IVIS) of 1.2

It was concluded that under the condition of this study, the test substance produced an IVIS score of 1.2 and therefore no categorisation for irritation could be made and the substance does not meet the criteria for classification in accordance with  UN GHS or EU CLP.

OECD492 (2019) - in vitro study was performed to assess the eye irritation potential of CPI-110A by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 μL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with CPI-110A. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (determined value for the test item 13.37%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, no prediction can be made for CPI-110A.

In silico Eye Irritation (QSAR)

The determination of eye irritation/corrosion potential of Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium  hexafluoroantimonate(1-) was performed by the Toxtree 3.1.0 and OECD QSAR Toolbox 4.3. The BfR rule-based decision support system classifies a compound as having the potential to cause adverse effects to the eye. All modelling was based on the input of SMILES notation for each constituent using SMILES descriptors. Descriptor values were assigned from the experimental information, which were for the purposes of the fulfilment of REACH (Regulation (EC) No. 1907, 2006), physical/chemical data requirements and from the structure of the chemical(s) itself.   When not available the values were predicted using OECD QSAR toolbox v4.3.

The assessment indicates that the prediction is suitable for the regulatory conclusion set out in section 4.1. This conclusion is with due regard to REACH Annex VI-XI.

The prediction is adequate for the Classification and Labelling and/or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006 in a weight of evidence approach and to aid in the fulfilment of the data requirements of Regulation (EC) No. 1907/2006: Annex VII.

Justification for classification or non-classification

The test item did not meet the criteria for classification for skin or eye irritation according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixture.