Bis(dodecylthio)dioctylstannane

Endpoint summary

Description of key information

Skin Corrosion

Under the conditions of the study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation

Under the conditions of the study, the test material is non-irritant in the in vitro skin irritation test.

Eye Irritation

Under the conditions of the study the test material had an IVIS of 0.8, therefore it is not irritating to the eye.

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:

07 September 2017 to 08 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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue lot number: 25840
- Delivery date: 05 September 2017

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT: 50 μL of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured or if it becomes coloured when in wet or aqueous conditions. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 50 μL of test material was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

MAIN TEST
PRE-INCUBATION
- The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-minute and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

APPLICATION OF TEST MATERIAL
- Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-minute exposure period was returned to the incubator, while the other was being dosed for the 60-minute exposure. For the 60-minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of the test material and 50 μL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-minute exposure period.
- When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure.

REMOVAL OF TEST MATERIAL AND CONTROLS AND MTT LOADING
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test material. 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. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
- After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

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

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

DATA EVALUATION
- Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.
The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material/ mean OD570 of the negative control) x 100
Classification of corrosivity potential is based on relative viabilities for both exposure times according to the following:
STEP 1:
< 50 % viability after 3 min exposure = corrosive
≥ 50 % viability after 3 min exposure and < 15 % viability after 60 min exposure = corrosive
≥ 50 % viability after 3 min exposure and ≥ 15 % viability after 60 min exposure = non-corrosive
STEP 2 for test materials identified as corrosive in step 1:
< 25 % viability after 3 min exposure = H314 Sub-category 1A
≥ 25 % viability after 3 min exposure = H314 Combination of sub-categories 1B-and-1C

- Quality Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
- Potassium hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-minute positive control is < 15 %.
- In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes of exposure and 1 hour of exposure

Duration of post-treatment incubation (if applicable):

Incubated for 3 hours with MTT

Number of replicates:

2 per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

95.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure period

Value:

97.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct MTT Reduction: The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

- Assessment of Colour Interference with the MTT endpoint: The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

- Test Material, Positive Control and Negative Control: Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.

- Quality Criteria:
The mean OD570 for the negative control treated tissues was 1.750 for the 3-minute exposure period and 1.746 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 3.2 % 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: Mean OD₅₇₀ Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue	Exposure Period	MeanOD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.788	1.750	0.054	3.1	100*
		1.711
	60 Minutes	1.815	1.746	0.098	5.6
		1.677
Positive Control	3 Minutes	0.069	0.065	0.006	na	3.7
		0.061
	60 Minutes	0.055	0.057	0.002	na	3.2
		0.058
Test Material	3 Minutes	1.719	1.665	0.077	4.6	95.1
		1.610
	60 Minutes	1.700	1.708	0.011	0.7	97.8
		1.716

OD = Optical density

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

na = Not applicable

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

The potential of the test material to cause skin corrosion was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions.

The corrosivity potential of the test material was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).  

The mean percentage viability of the test material treated tissues was 95.1 and 97.8 % after 3 and 60 minute exposure times, respectively. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 2017 to 27 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Source: SkinEthic Laboratories, Lyon, France
- Tissue lot number: 17-EKIN-047
- Date received: 21 November 2017

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test material is checked for the ability to directly reduce MTT: 10 µL of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test material turns blue/purple, the test material is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 10 µL of test material was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
- Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert, whether the tissues were satisfactory, whether the temperature indicator colour was satisfactory and whether the agar medium colour was satisfactory.
- 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labelled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

APPLICATION OF THE TEST MATERIAL (DAY 1)
- 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.
- Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm²) of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7 minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the exposure period, each tissue was removed from the well using forceps and 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 material. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.

MTT LOADING/ FORMAZAN EXTRACTION (DAY 3)
- Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.
- 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5 % CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

NUMBER OF REPLICATE TISSUES: 3

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.

DATA EVALUATION
- Quantitative MTT Assessment (Percentage Tissue Viability) :
For the test material the relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material/ mean OD570 of the negative control) x 100
Classification of irritation potential is based upon relative mean tissue viability following the 15 minute exposure period followed by the 42 hour post exposure incubation period according to the following:
A relative mean tissue viability ≤ 50 % = Irritant (H314 or H315 Category 1 or 2)
A relative mean tissue viability > 50 % = Non- irritant (Not classified for irritation)

- Quality Criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
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 OD570 for the negative control treated tissues is ≥ 0.6 and ≤ 1.5, and the SD value of the percentage viability is ≤ 18 %.
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 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL (26.3 µL/cm²)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5 % w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct MTT Reduction:
The MTT solution containing the test material did not turn blue or purple which indicated that the test material did not directly reduce MTT.

- Assessment of Colour Interference with the MTT endpoint:
The solution containing the test material was colourless. It was therefore unnecessary to run colour correction tissues.

- Test Material, Positive Control and Negative Control:
The individual and mean OD570 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.
The relative mean viability of the test material treated tissues was 98.1 % after a 15 minute exposure period and 42 hour post exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

- Quality Criteria:
The test was repeated due to a failure to meet the assay acceptance criteria. The relative mean tissue viability for the positive control treated tissues was 6.0 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.8 %. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.855 and the standard deviation value of the viability was 6.6 %. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 9.7 %. The test material acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₇₀Values and Viabilities for the Negative Control, Positive Control and Test Material

Treatment	OD570 of tissues	Mean OD570 of triplicate tissues	± SDof OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.821	0.855	0.056	96.0	100*	6.6
	0.920			107.6
	0.824			96.4
Positive Control	0.045	0.051	0.016	5.3	6.0	1.8
	0.040			4.7
	0.069			8.1
Test Material	0.913	0.839	0.083	106.8	98.1	9.7
	0.749			87.6
	0.854			99.9

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Executive summary:

An in vitro skin irritation test using a human skin model was carried out in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

The skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. Triplicate tissues were treated with the test material 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-labelled 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-labelled 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 material treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was 98.1 % after the 15 minute exposure period and 42 hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

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:

07 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

yes

Remarks:

(please refer to "Principles of method if other than guideline" for further information)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(please refer to "Principles of method if other than guideline" for further information)

Principles of method if other than guideline:

The positive control group had an overall IVIS of 60.3, which was marginally higher than the criteria range set for an acceptable test. As the score was only marginally exceeded, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.

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: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes followed by 90 minutes with sodium fluorecein

Number of animals or in vitro replicates:

3 replicates for each condition (test material, negative control and positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

TREATMENT METHOD
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.

REMOVAL OF TEST SUBSTANCE
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

SODIUM FLUORESCEIN
Following the final 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 (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. After sodium fluorescein incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The corneal opacity readings were taken using a calibrated opacitometer. The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled 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)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

DATA INTERPRETATION
The test material was classified according to the following:
- IVIS ≤ 3 = Not classified for irritation
- IVIS > 3; ≤ 55 = No prediction can be made
- IVIS > 55 = Category 1, H318: Causes serious eye damage

ACCEPTABILITY OF THE ASSAY
For an acceptable test the following control criteria should be achieved:
- The test was acceptable if the positive control produced an IVIS which fell within two standard deviations of the historical mean collated during 2016 for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 31.6 to 58.7.
- The test was acceptable if the negative control produced an IVIS which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 3.0 and for permeability ≤ 0.077.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- A summary of the results of the study can be seen in Table 1.
- The corneas treated with the test material were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF THE RESULTS
- The positive control In Vitro Irritancy Score was above the range of 31.6 to 58.7 (60.3). The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
- The negative control gave opacity of ≤ 3.0 and permeability ≤ 0.077. The negative control acceptance criteria were therefore satisfied.

Table 1: Summary of opacity, permeability and in vitro irritancy scores

Treatment	Mean Opacity	Mean Permeability	Mean In Vitro Irritancy Score
Negative control	0.0	0.002	0.0
Positive control	32.3	1.862	60.3
Test material	0.3	0.034	0.8

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study the test material had an IVIS of 0.8, therefore it is not irritating to the eye.

Executive summary:

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 using the Bovine Corneal Opacity and Permeability (BCOP) test method, under GLP conditions.

The test was performed to assess damage by the test material using quantitative measurements of changes in corneal opacity and permeability.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control materials 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). 

The positive control In Vitro Irritancy Score was above the range of 31.6 to 58.7 (60.3). The positive control acceptance criterion was therefore not satisfied and this is reported as a deviation. The negative control gave opacity of ≤ 3.0 and permeability ≤ 0.077 and the negative control acceptance criteria were therefore satisfied.

Under the conditions of this study the test material had an IVIS of 0.8, therefore it is not irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion

The potential of the test material to cause skin corrosion was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The corrosivity potential of the test material was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).  

The mean percentage viability of the test material treated tissues was 95.1 and 97.8 % after 3 and 60 minute exposure times, respectively. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation

An in vitro skin irritation test using a human skin model was carried out in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. Triplicate tissues were treated with the test material 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-labelled 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-labelled 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 material treated tissues relative to negative control tissues).

The relative mean viability of the test material treated tissues was 98.1 % after the 15 minute exposure period and 42 Hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Eye Irritation

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 using the Bovine Corneal Opacity and Permeability (BCOP) test method under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test was performed to assess damage by the test material using quantitative measurements of changes in corneal opacity and permeability.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control materials 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). 

The positive control In Vitro Irritancy Score was above the range of 31.6 to 58.7 (60.3). The positive control acceptance criterion was therefore not satisfied and this is reported as a deviation. The negative control gave opacity of ≤ 3.0 and permeability ≤ 0.077 and the negative control acceptance criteria were therefore satisfied.

Under the conditions of this study the test material had an IVIS of 0.8, therefore it is not irritating to the eye.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin and eye corrosion or irritation.