Tetrabutyltin

Endpoint summary

Description of key information

Skin Corrosivity: The purpose of this test was to evaluate the corrosivity potential of the test material using the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

Skin irritancy: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstituted human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The test material was considered to be Irritant.

Eye irritancy: The purpose of the study was to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. Following assessment of the data, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

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:

28th to 30th April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstituted Human Epidermis (RHE)

Details on animal used as source of test system:

The EPISKIN model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit 0.38 cm2
Supplier: SkinEthic Laboratories, Nice, France
Date received: 27 April 2010

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST
ASSESSMENT OF DIRECT TEST MATERIAL REDUCTION OF MTT
- MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by the procedure described below.

- Test for Direct MTT Reduction:
As specified, 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 according to the procedure below:
50 µL of the test material was added to 2.2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used 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.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
2.2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12 well plate was used for each test material, control and time point. The tissues were incubated at 37 °C, 5 % CO2 in air for at least 24 hours. After 24 hours the medium underneath the tissues was refreshed and tissues were returned to the incubator for a further 24 hours.

MAIN TEST
APPLICATION OF TEST MATERIAL AND RINSING (DAY 2)
2.2 mL of assay medium, warmed to approximately 37 °C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control materials for an exposure period of 240 minutes. 50 µL of the test material was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 µL of 0.9 % w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of glacial acetic acid served as positive controls. The treated tissues were kept in the biological safety cabinet at room temperature for the appropriate exposure period.
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
2.2 mL of 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ± 5 minutes at room temperature in a biological safety cabinet ensuring that the plates were protected from light. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability). Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was taken using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 850 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 3)
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 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of the test material was applied

Duration of treatment / exposure:

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes.

Duration of post-treatment incubation (if applicable):

3, 60 and 240 minutes.

Number of replicates:

Two per time point

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 3 minutes

Value:

99

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 60 minutes

Value:

108.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 240 minutes

Value:

92.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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.

TEST MATERIAL, POSITIVE CONTROL MATERIAL AND NEGATIVE CONTROL MATERIAL:
Mean OD540 values and viabilities for the negative control, positive control and test material are given in Table 1.
The relative mean viability of the test material treated tissues was as follows:
240 minutes of exposure: 92.8 %
60 minutes of exposure: 108.7 %
3 minutes of exposure: 99.0 %

The qualitative evaluation of tissue viability is given in Table 2.
Following the 3, 60 and 240-minute exposure periods, the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

QUALITY CRITERIA
The relative mean tissue viability for the positive control treated tissues was 4.8 % relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

Table1: Mean OD540 Values and Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	Exposure Period (minutes)	Mean OD540 of duplicate tissues	Relative mean viability (%)
Negative Control Material	240	0.207	100*
Positive Control Material	240	0.010	4.8
Test Material	240	0.192	92.8
	60	0.225	108.7
	3	0.205	99.0

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

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Exposure Period (minutes)	Tissue 1	Tissue 2
Negative Control Material	240	-	-
Positive Control Material	240	++	++
Test Material	240	-	-
	60	-	-
	3	-	-

MTT Visual Scoring Scheme of EpiSkin Tissues

-     =  Blue tissue (viable)

+    =  Blue/white tissue (semi-viable)

++  =  Tissue completely white (dead)

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of the test material using the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

The EPISKIN model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) chemicals.

Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes. 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 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 540 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 were as follows:

240 minutes of exposure : 92.8 %

60 minutes of exposure : 108.7 %

3 minutes of exposure : 99.0 %

The quality criteria required for acceptance of results in the test were satisfied. The test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th May to 1st June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Validated in vitro method following the EPISKIN reconstituted human epidermis model

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstituted Human Epidermis (RHE)

Details on animal used as source of test system:

The EPISKIN model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN™ MODEL KIT
Date received: 26 May 2010

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST
ASSESSMENT OF DIRECT TEST MATERIAL REDUCTION OF MTT
- MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test substance with MTT. A test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test substance is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test substance present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

- Test for Direct MTT Reduction
As specified, a test substance 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, each test substance is checked for the ability to directly reduce MTT according to the following procedure:
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 % CO2in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
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 for at least 24 hours.

MAIN TEST
APPLICATION OF TEST MATERIAL AND RINSING (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 of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 µL of PBS 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 7 minutes of 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. The plate(s) were kept in the biological safety cabinet at room temperature for approximately 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS 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 approximately 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 approximately 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 plate(s). 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. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual Scoring scheme. Following qualitative evaluation of tissue viability, 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.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6)
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 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL MATERIALS, MTT AND ACIDIFIED ISOPROPANOL
The negative control material was used as supplied.
The positive control material was prepared as a 5 % w/v aqueous dilution.
A 3 mg/mL MTT stock solution was prepared in PBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N concentration of hydrochloric acid in Isopropanol was prepared when required.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL of the test material was applied to the epidermis surface.

Duration of treatment / exposure:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes

Duration of post-treatment incubation (if applicable):

A post exposure incubation period of 42 hours

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean viability (%)

Run / experiment:

Time point: 15 minutes

Value:

38.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

TEST MATERIAL, POSITIVE CONTROL MATERIAL AND NEGATIVE CONTROL MATERIAL
The individual and mean OD540 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 38.5 % after a 15 Minute exposure period.

The qualitative evaluation of tissue viability is given in Table 2.
Following the 15-Minute exposure period the test material treated tissues appeared blue/white which was considered indicative of semi viable tissue.

The test material was considered to be Irritant (I).

QUALITY CRITERIA
The relative mean tissue viability for the positive control treated tissues was ≤40 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤20 %. The positive control acceptance criterion was therefore satisfied.
The mean OD540 for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤20 %. The negative control acceptance criterion was therefore satisfied.

Table1: Mean OD540 Values and Percentage Viabilities for the Negative Control, Positive Control and Test Material

Material	OD540 of tissues	Mean OD540 of triplicate tissues	±SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Material	0.732	0.772	0.082	94.8	100*	10.6
	0.718			93.0
	0.866			112.2
Positive Control Material	0.049	0.050	0.009	6.3	6.5	1.2
	0.042			5.4
	0.060			7.8
Test Material	0.253	0.297	0.057	32.8	38.5	7.4
	0.361			46.8
	0.277			35.9

SD = Standard deviation

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

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	+	+	+

MTT visual scoring scheme

- = blue tissue (viable)

+ = blue/white tissue (semi-viable)

++ = tissue is completely white (dead)

INTERPRETATION OF RESULTS

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 OD540 of test material/mean OD540 of negative control) x 100

Classification of irritation potential is based upon relative tissue viability following the 15 Minute exposure period followed by the 42 Hour post-exposure incubation period according to the following table:

Criteria for in vitro interpretation	Classification
Mean tissue viability is ≤50 %	Irritant (I) R38
Mean tissue viability is >50 %	Non-Irritant (NI)*

*The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation will be determined for test materials which are found to be borderline non-irritant based upon the MTT cell viability endpoint (mean tissue viability 51 to 60 %). This complimentary end point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test material was considered to be an irritant.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstituted human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end point will be used to either confirm a non-irritant result or will be used to override the non irritant result.

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 approximately 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 540 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 38.5 % after the 15 minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

The test material was considered to be an irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

30th June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

no guideline available

Principles of method if other than guideline:

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

The test material was applied as evenly as possible to the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C).

Observation period (in vivo):

Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment

Number of animals or in vitro replicates:

Three eyes were treated with test material, two additional eyes remained untreated for control purposes.

Details on study design:

PRE-TEST PROCEDURES
SUPERFUSION CHAMBER
The water heating circulator (Julabo MP5, Jencons (Scientific) Ltd., Leighton Buzzard, Beds, UK), was adjusted so that the temperature of the water flowing through the water jacket of the superfusion apparatus, gave a stable temperature, of 32 ± 1.5 °C, within the chambers of the apparatus. A peristaltic pump (205S/BA, Watson Marlow Ltd, Falmouth, Cornwall; UK) was used to supply saline solution at a flow rate of 0.15 to 0.4 mL/minute (at approximately 30 °C) into the rear of each chamber of the apparatus in order to irrigate the surface of the cornea.

SELECTION OF EYES
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1 % w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope (Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes.

ENUCLEATION OF EYES
The donor rabbits were sacrificed by intravenous administration of an overdose of sodium pentobarbitone. Immediately afterwards, two to three drops of saline solution (approximately 32 °C) were applied to the cornea to prevent desiccation during excision. The eye was then carefully removed, positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber adjusted so that saline solution was allowed to irrigate the surface of the cornea. The eyes were then allowed to equilibrate for approximately thirty minutes. Following the equilibration period, the eyes were re-examined to ensure they had not been damaged during excision. Corneal thickness was also measured using the ultrasonic pachymeter. Any eyes in which the corneal swelling was greater than 10 % relative to the pre-enucleation measurement, or in which the cornea was stained with fluorescein, were rejected. The post equilibration corneal thickness values for each eye were recorded.

MAIN TEST PROCEDURE
TEST MATERIAL ADMINISTRATION
Three eyes were treated with test material, two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.
The test material was used undiluted as supplied. A volume of 0.1 mL of the test material was applied as evenly as possible to the surface of the cornea.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C). Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye. The untreated eyes were similarly washed and used for control purposes.
- Time after start of exposure: 10 seconds

SCORING SYSTEM:

CORNEA
The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. Severity of corneal cloudiness is graded as follows:
0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stroma.
1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit-lamp. The underlying
structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse illumination, underlying structures are clearly visible.
3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination the underlying structures are just visible.
4 = Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With diffuse illumination, the underlying structures cannot be seen.

The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4.
0 = Normal cornea with no area of cloudiness
1 = 1 to 25 % area of stromal cloudiness
2 = 26 to 50 % area of stromal cloudiness
3 = 51 to 75 % area of stromal cloudiness
4 = 76 to 100 % area of stromal cloudiness

FLUORESCEIN
The use of fluorescein is a valuable aid in defining epithelial damage for fluorescein staining. The area can be judged as a 0 to 4 scale using the same terminology as for corneal cloudiness. The intensity of fluorescein staining can be divided into a 0 to 4 scale.
0 = Absence of fluorescein staining
1 = Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but are not completely obliterated.
4 = Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be seen.

REFERENCE:
Hackett R B and McDonald T O, Eye Irritation. In: Advances in Modern Toxicology: Dermatoxicology. 4th ed. (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749 815.

TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.

Irritation parameter:

cornea opacity score

Run / experiment:

Time point 240 minutes

Value:

3

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: corneal swelling

Run / experiment:

Time point 60 minutes

Value:

13

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: corneal swelling

Run / experiment:

Time point 120 minutes

Value:

18.4

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: corneal swelling

Run / experiment:

Time point 240 minutes

Value:

26.9

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

other: Fluoroscein uptake

Run / experiment:

Time point 240 minuted

Value:

3

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

CORNEAL OPACITY
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study period.

CORNEAL THICKNESS
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3.
Corneal swelling of the test eyes during the study period was considerably greater than that observed in the control eyes over the same period.

CORNEAL CONDITION
The condition of the corneal epithelium following treatment is given in Table 4.
Sloughing of the cornea was noted in two test eyes. One test eye and the control eyes appeared normal during the study period.

FLUORESCEIN UPTAKE
Individual scores for fluorescein uptake are given in Table 5.
Slight fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Please refer to illustration for tabulated results (Tables 1 -5)

Interpretation of Results

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*	REET Cut‑Off Value
Maximum Corneal Opacity (Corneal Cloudiness x Area)	> or = 4
Maximum Fluorescein Uptake (Intensity x Area)	> or = 4
Mean Corneal Swelling (mins): 60, 120, 240	> or = 25 %
Corneal Epithelium Observations	Any with pitting, mottling or sloughing

*Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

((mean corneal thickness post-treatment) – (mean corneal thickness post equilibration) /(mean corneal thickness post equilibration)) x 100

- A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 minute post treatment observation periods.

- A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

Executive summary:

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. 

0.1 mL of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ± 1.5 °C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9 % Sodium Chloride).

Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal Opacity	Fluorescein Uptake	Corneal Swelling (%)						Condition of Corneal Epithelium
		Test Eyes*			Control Eyes**
Cloudy x Area	Int x Area	60 mins	120 mins	240 mins	60 mins	120 mins	240 mins
3	3	13.0	18.4	26.9+	5.1	5.1	3.5	Sloughing+

Following assessment of the data for all endpoints the test material was considered to have the potential to cause severe ocular irritancy in vivo.

*For each time point the swelling recorded is the mean of three eyes

**For each time point the swelling recorded is the mean of two eyes

Int = Intensity of fluorescein uptake

+ = Meets or exceeds cut-off value indicating a severe ocular irritant

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

There are two key in vitro studies for this endpoint.

- The first study (Warren, 2010) assesses skin corrosivity designed to meet the requirements of the OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004). A reliability rating of 1 was assigned according to the criteria of Klimisch et al. (1997) as this was conducted to a guideline and under GLP conditions.

Under the conditions of this study, the test material was considered to be Non-Corrosive to the skin and accredited the EU risk phrase of No label and a UN packing group Non-Corrosive.

- The second study (Warren, 2010) assesses skin irritancy according to a validated in vitro method following the EPISKIN™ reconstituted human epidermis model. A reliability rating of 1 was assigned according to the criteria of Klimisch et al. (1997) as this was conducted to a validated in vitro method and under GLP conditions.

Under the conditions of this study, the in vitro data indicates that the substance is considered irritating to the skin.

Eye

One key study is available for the assessment of this endpoint (Sanders, 2010). The study was performed in accordance with a validated in vitro method using enucleated rabbit eyes.

A reliability score of 1 was assigned to the study according to the criteria outlined in Klimisch et al. (1997), as this was conducted to a validated method under GLP conditions.

Under the conditions of this study, following assessment of the in vitro data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

Justification for classification or non-classification

In accordance with the criteria for classification as set forth in Regulation (EC) No 1272/2008, the substance requires classification as a Category 2 skin irritant (H315: Causes skin irritation) and Category 1 for eye damage (H318: Causes serious eye damage).