Selenium disulphide

Endpoint summary

Description of key information

Skin Corrosion - In Vitro

Not corrosive in the Reconstructed Human Epidermis (RHE) Test

Skin Irritation - In Vitro

Non irritant in the human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)).

Eye Irritation - In Vitro

No induction of ocular irritation in the ICE test.

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:

From 28 September 2017 to 29 September 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:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Selenium Disulphide
CAS Number: 7488-56-4
Batch number: PMC / 285 / 16
Appearance: Orange/yellow powder
Expiry date: 24 November 2019
Purity: 100%
Storage conditions: Room temperature (between 5oC and 30oC), protected from light Safety precautions: Enhanced safety precautions were applied considering the supplied safety data sheet to assure personnel health and safety.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

not specified

Details on animal used as source of test system:

Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-039, Expiry Date: 02 October 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Quality Control
EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study).

Number of Replicate Wells
In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Kit Reception
In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 27 September 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

Acidified isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

Control samples:

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

- 20 mg of test item was applied evenly to the epidermal surface of each of two test item treated skin units and each additional control skin units and then 100 μL
physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

4 hours (+/- 10 minutes)

Duration of post-treatment incubation (if applicable):

3 hours (+/- 15 minutes)

Number of replicates:

2 replicates per test or control (positive and negative)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Selenium Disulphide Run 1

Value:

91.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Selenium Disulphide Run 2

Value:

94.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Selenium Disulphide Mean

Value:

92.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.002, Non Specific Colour% (NSCliving%) was calculated as 0.3%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional
controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2. The mean OD value for
the test item treated skin samples showed an 92.7% relative viability compared to the negative control.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.758).
The two positive control treated tissues showed 1.0 % viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 3.1%.
The difference of viability between the two negative control tissue samples in the MTT assay was 0.9%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

Additional control	Optical density (OD)			NSC% (living)
Treated with selenium disulphide	Number	Measured	Blank corrected
	1	0.049	0.002	0.3
	2	0.045	-0.001*
	Mean	--	0.002

Notes:

1. Mean blank value was 0.046.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. * OD value was excluded from the corrected calculation (negative value).

 

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical density (OD)			Viability (% RV)
	Number	Measured	Blank corrected
Negative control (physiological saline 0.9 %(w/v) NaCl	1	0.808	0.761	100.4
	2	0.801	0.755	99.6
	Mean	--	0.758	100.0
Positive control Glacial acetic acid	1	0.054	0.008	1.0
	2	0.054	0.008	1.0
	Mean	--	0.008	1.0
Selenium disulphide	1	0.738	0.692	91.3
	2	0.760	0.713	94.1
	Mean	--	0.702	92.7

Notes:

1. Mean blank value was 0.046.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

HISTORICAL CONTROL DATA

	Negative control (Physiological saline)	Positive control (Glacial acetic acid)
Minimum optical density (OD)	0.611	0.005
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.871	0.017
Standard Deviation (SD)	0.164	0.010
Number of cases	81	81

 

Note: All optical density (OD) values measured are background corrected values (measured at 570±30 nm)

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Selenium Disulphide, the mean cell viability was 92.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of Selenium Disulphide test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with Selenium Disulphide test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Selenium Disulphide, the mean cell viability was 92.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in thisin vitroEPISKIN™ (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 September 2017 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009)

Version / remarks:

ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Selenium Disulphide
CAS Number: 7488-56-4
Batch number: PMC / 285 / 16
Appearance: Orange/yellow powder
Expiry date: 24 November 2019
Purity: 100%
Storage conditions: Room temperature (between 5oC and 30oC), protected from light Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study [10] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-038, Expiry Date: 25 September 2017) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994) [7]. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Kit Contents
Units: EPISKINTM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM (SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 17 MAIN3 040; Exp. Date: 27 September 2017)
A flask of sterile “Assay Medium” (Batch No.: 17 ESSC 037; Exp. Date: 27 September 2017)

Quality Control
EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories.

Number of Replicate Wells
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM (SM) kit was kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of
the test.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 19 September 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

Acidified Isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was
applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement.

Check-method for possible direct MTT reduction with test item
10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube.
Thus, if the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water* and/or extracting solution (e.g. acidified isopropanol) (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
*Note: Water is the environment during exposure.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

Control samples:

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

42 hours

Duration of post-treatment incubation (if applicable):

3 hours (± 5 minutes)

Number of replicates:

3 replicates per group (test item, positive control and negative control)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

96.1

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:

2

Value:

98.2

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:

3

Value:

94.8

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:

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.829). Standard deviation of the viability results for negative control samples
was 0.2.

The positive control treated tissues showed 3.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive
control samples was 0.5.

Note: The OD data of two replicate tissues and the mean OD data at the positive control in this study was lower than the minimum OD of the historical control range (0.032). This fact has no impact on the results or integrity of the study since the positive control material showed severe effect.

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 1.7.

The mean OD value of the blank samples (acidified isopropanol) was 0.047. All these parameters met the acceptability criteria, therefore the study was considered to be valid.

ADDITIONAL CONTROLS

As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded. As the test item was coloured, two additional test item-treated living tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.010, Non Specific Colour % was calculated as 1.2% . This value was below 5%, therefore additional data calculation was not necessary.

 

Additional control	Optical density (OD)			NSC%
		Measured	Blank corrected
Treated with selenium disulphide	1	0.051	0.004	1.2%
	2	0.062	0.016
	Mean	--	0.010

 

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative control (phosphate buffer saline)	1	0.876	0.829	100.0
	2	0.877	0.830	100.2
	3	0.874	0.827	99.8
	Mean	--	0.829	100.0
Positive control 5% SDS solution	1	0.074	0.027	3.3
	2	0.080	0.033	4.0
	3	0.073	0.026	3.1
	Mean	--	0.029	3.5
Test item selenium disulphide	1	0.843	0.797	96.1
	2	0.861	0.814	98.2
	3	0.861	0.786	94.8
	Mean	--	0.799	96.4

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

HISTORICAL CONTROL DATA

 

	Negative control (PBS)	Positive control (5% (w/v) SDS solution)
Mean optical density (OD)	0.802	0.094
Standard deviation	0.157	0.048
Minimum optical density (OD)	0.573	0.032
Maximum optical density (OD)	1.362	0.354
Number of cases	111	102

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure to Selenium Disulphide, the mean cell viability was 96.4% compared to the negative control. This is above the threshold of 50%, therefore the test
item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-irritant to skin.

Executive summary:

An in vitro skin irritation test of Selenium Disulphide test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring their cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline [1].

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure to the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure to Selenium Disulphide, the mean cell viability was 96.4% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 September 2017 to 27 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Selenium Disulphide
CAS Number: 7488-56-4
Batch number: PMC / 285 / 16
Appearance: Orange/yellow powder
Expiry date: 24 November 2019
Purity: 100%
Storage conditions: Room temperature (between 5oC and 30oC), protected from light
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)

Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within 2 hours of collection in each experiment.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

2 replicates per group (test material, positive control, negative control)

Details on study design:

SELECTION AND PREPARATION OF EYES FOR THE TEST
Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly.

The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.
 
Identification
The eyes were identified by chamber number, marked on the door of the chamber.

The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.

In each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.

One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

Test item removal
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed in the experiments. The test item treated eyes were rinsed additional gentle rinsing with 20 mL saline after treatment in Experiment II.
Note: Physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 72034Y05-1,
Expiry date: 30 April 2020) was used for rinsing.

Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Retention of chicken’s eyes
At the end of the procedure, the corneas of the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: Reanal, Batch number: KTM20001, Expiry date: March 2019) was used for potential histopathology and stored at room temperature.

Classification criteria:
The following criteria is used to identify the probably eye irritancy potential of test items. In the case where the result indicates Non-irritant or Corrosive/Severely Irritating, then the test item can be classified. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.

UN GHS Classification Combinations of the three ICE Classes
No Category 3×I
2×I, 1×II

No prediction can be made Other combinations
Category 1 3×IV
2×IV, 1×III
2×IV, 1×II*
2×IV, 1×I*
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of epithelium (in at least 1 eye)

Remark:*: combinations of categories less likely to occur

Irritation parameter:

percent corneal swelling

Remarks:

75 - 240 min

Run / experiment:

1

Value:

>= 1.1 - <= 1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

0.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

2

Value:

1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

2

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in each experiment.
This study was considered to be valid.

The test item Selenium Disulphide showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes with Selenium Disulphide, the test item was non-irritant, UN GHS Classification: No Category.

In experiment II. test item was stuck on all cornea surfaces after the post-treatment rinse, all cornea surfaces were cleared at 30 minutes after the post-treatment rinse. In each experiment positive control material was stuck on all cornea surfaces after the posttreatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

No other morphological effect was observed in the study.

Test Item Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.1 %	I
Mean maximum corneal swelling at up to 240 min	1.6 %	I
Mean maximum corneal opacity	0.17	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

Test Item Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.6 %	I
Mean maximum corneal swelling at up to 240 min	1.6 %	I
Mean maximum corneal opacity	0.50	I
Mean fluorescein retention	0.00	I
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xI

 

Positive Control Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10.9%	II
Mean maximum corneal swelling at up to 240 min	25.1%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck onallcornea surfaces after the post- treatment rinse.The cornea surfaces (3/3) were not clearedat 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

Positive Control Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10.3%	II
Mean maximum corneal swelling at up to 240 min	25.5%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post- treatment rinse.The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

The positive control Imidazole was classified as severely irritating, UN GHS Classification:Category 1.

 

Negative Control Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

Negative Control Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the in vitro eye irritation assays in isolated chicken eyes with Selenium Disulphide, it was concluded that the test item was non-irritant.
UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes  were  treated  with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on one eye. No fluorescein retention change was noted on three eyes.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. No fluorescein retention change was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on the in vitro eye irritation assays in isolated chicken  eyes  with  Selenium Disulphide, it was concluded that the test item was non-irritant, UN GHS Classification: No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion - In Vitro

This study was designed to be compatible with the following:

-OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method” (adopted 28 July 2015)

-Method B.40 of Commission Regulation (EC) No. 440/2008

An in vitro skin corrosivity test of Selenium Disulphide test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with Selenium Disulphide test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Selenium Disulphide, the mean cell viability was 92.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-corrosive to the skin.

 

Skin Irritation – In vitro

This study was designed to be compatible with the following:

-OECD Guidelines No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (28 July 2015)

-Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “In Vitro Skin Irritation Reconstructed Human Epidermis Model Test”, amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

-EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

An in vitro skin irritation test of Selenium Disulphide test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring their cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-l)-2,5 -diphenyltetrazolium bromide)) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline [1].

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure to the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure to Selenium Disulphide, the mean cell viability was 96.4% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-irritant to skin.

 

Eye Irritation – In vitro

This study was designed to be compatible with the following:

Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage: OECD Guidelines for the Testing of Chemicals No. 438 (26 July 2013)

 

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

 

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on one eye. No fluorescein retention change was noted on three eyes.

 

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. No fluorescein retention change was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

 

Based on these in vitro eye irritation assays in isolated chicken eyes with Selenium Disulphide, the test item was non-irritant, UN GHS Classification: No Category.

 

Justification for classification or non-classification

Skin Corrosion

Based on the results, selenium disulphide does not have to be classified and has no obligatory labelling requirement for skin corrosivity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

 

Skin Irritation

Based on the results, selenium disulphide does not have to be classified and has no obligatory labelling requirement for skin irritantion according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

 

Eye Irritation

Based on the results, selenium disulphide does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).