Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Endpoint summary

Description of key information

SKIN
Not irritating (in vitro); EPISKIN-SM model
EYE
Not irritating (in vitro); isolated chicken eyes

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2015 to 14 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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

Principles of method if other than guideline:

Due to the interfering colour of the test material (dark blue) the plate was also measured at 620 nm in order to more precisely evaluate the possible amount of test material remaining on the surface on the skin units and the resulting non-specific colour. This fact was considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN-SM model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Model: EPISKIN-SM. The EPISKIN-SM is 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 a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-032, Expiry Date: 17 August 2015
- 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 = not acceptable). The colour of the temperature indicator was inspected to verify that the kit had 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 = good; grey or black = not acceptable). The kits were found to be in good order at reception.
- Storage: The EPISKIN-SM kit was kept in the packaging at 37 °C; the Assay Medium and Maintenance Medium supplied with the kits were stored at 2 to 8 °C until the initiation of the test.

KIT CONTENTS
- Units: EPISKIN-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: EPISKIN-SM biopsy punch for easy sampling of epidermis
- Medium: A flask of sterile “Maintenance Medium”, Batch No.: 15 MAIN3 032; Exp. Date: 19 August 2015. A flask of sterile “Assay Medium”, Batch No.: 15 ESSC 032; Exp. Date: 19 August 2015. A flask of sterile “Assay Medium”, Batch No.: 15 ESSC 031; Exp. Date: 12 August 2015.

KILLED EPIDERMIS
For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 15-EKIN-022, Expiry Date: 08 June 2015) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO₂, in a >95 % humidified atmosphere for 48 hours (± 1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 06 June 2015. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

Type of coverage:

open

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Tissues were exposed to both positive (5 % (w/v) aqueous sodium dodecyl sulphate in distilled water) and negative (phosphate buffered saline) concurrent controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (test material was applied in its original form, though ground to a fine powder before use)

Duration of treatment / exposure:

15 minutes

Observation period:

After exposure the skin tissue was thoroughly rinsed to remove the test material, followed by incubation for 42 hours.

Number of animals:

Three replicates were used for the test material. Five negative controls and three positive controls were also run in the assay. As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. Furthermore, as the test material had an MTT interacting potential, three additional test material-treated killed epidermis and three negative control treated killed epidermis were used in the study.

Details on study design:

PERFORMANCE OF THE STUDY
- Pre-incubation (Day -1)
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO₂, in a >95 % humidified atmosphere.

- Application and rinsing (day 0)
Test Material: 10 μL of distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis, and then 10 mg of test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a curved flat spatula.
Positive and negative control: 50 μL of positive control (5 % (w/v) SDS solution) or negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to evenly cover the entire epidermal surface.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (24.7 to 25.9 °C).
The test material was checked for MTT interacting potential and was found to be an MTT-interacting substance; therefore in addition to the normal procedure, three test material treated killed epidermis and three negative control treated killed epidermis were used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The same treatment steps were followed in case of the killed tissues as in case of the living tissues.
After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1 h) at 37 °C in an incubator with 5 % CO₂.

- MTT test (Day 2)
After the 42 hour incubation, all EPISKIN-SM units (except one colour control unit) were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 minutes) at 37 °C in an incubator with 5 % CO₂ protected from light.

- Formazan extraction (Day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (0.04 N HCl); one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

- Cell viability measurements (Day 2)
Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate. The OD (optical density or absorbance) of the samples were measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL /well) was used as blank.
In order to get more information about the amount of test material that remained on the surface of the skin units, the plate was measured additionally at 620 nm (the test material has a dark blue colour, so these wavelengths were considered to be suitable for this purpose).
The proper status of the instrument was verified by measuring a Verification plate (Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14) at the required wavelength on each day before use. Furthermore, the absorption spectrum of the test material was taken by a U-2900 spectrophotometer.

Irritation / corrosion parameter:

other: other: Relative viability percentage

Value:

85.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: n/a. Remarks: The OD values for the test material treated skin samples showed 85.9 % relative viability.. (migrated information)

Irritant / corrosive response data:

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 1. The OD values for the test material treated skin samples showed 85.9 % relative viability.

VALIDITY OF THE TEST
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 (0.794). Standard deviation for negative control samples was 7.0.
The positive control treated tissues showed 7.2 % viability demonstrating the proper performance of the assay. The standard deviation value for positive control samples was 1.4.
The standard deviation for viability values of the three test material-treated tissue samples in the MTT assay was 9.8.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Other effects:

ADDITIONAL CONTROLS
As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. The optical density (measured at 570 nm) of this tissue was 0.105, Non Specific Colour % was calculated as 13.2%. This value was above 5 %, therefore additional data calculation was necessary.
As colour change (blue colour) was observed after three hours of incubation of the test material in MTT working solution, the test material might interact with MTT. Therefore, additional controls and data calculations were used to exclude the false estimation of viability. Based on these observed OD (0.266), the calculated NSMTT (non-specific MTT reduction) was 33.7 %.
However, the observed colour was the test material itself and it did not derive from non-specific MTT reduction based on the absorption spectra of the test material. Therefore, this value was not used for correction

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Treatment	Optical Density (OD)			TODTT	Viability (%)
	Replicate	Measured	Blank Corrected
Negative Control (PBS)	1	0.860	0.814		103.0
	2	0.804	0.758		95.9
	3	0.778	0.731		92.6
	4	0.919	0.873		110.5
	5	0.820	0.773		97.9
	Mean		0.794		100
Positive Control (5 % w/v SDS)	1	0.095	0.049		6.1
	2	0.116	0.070		8.8
	3	0.099	0.052		6.6
	Mean		0.057		7.1
Test Material	1	0.743	0.697	0.592	75.0
	2	0.853	0.807	0.702	88.9
	3	0.892	0.845	0.741	93.8
	Mean		0.783	0.678	85.9

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample.

3. TODTT: An appropriate correction for non-specific colour (subtracting the NSC value of 0.105 was applied for test material treated samples.

4. Based on the absorbance values measured at 620 nm, no significant amount of test material remained on the test material treated skin units (values in the 0.372 to 0.457 range compared to the 0.360 to 0.432 values of the negative control samples).

5. Negative control replicates #4 and #5 were washed three times.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material was determined to be non irritating to the skin.

Executive summary:

A study was performed in vitro to assess the irritancy potential of the test material in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

The in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Disks of EPISKIN (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material 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 (five replicates) and positive controls (three replicates), respectively.

An additional disk was used to provide an estimate of colour contribution from the test material. Furthermore, three additional test material treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test material. 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 material is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 85.9 % compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin under the conditions of this study. The experiment met the validity criteria, therefore the study was considered to be valid.

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:

12 August 2015 to 25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according 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)

Species:

other: isolated chicken eyes

Strain:

other: ROSS 308 and COBB 500

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. 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 to 5 heads per box) and transported to the test site at ambient temperature. The heads were transported at the earliest convenience and processed within approximately 2 hours after receipt.
The ROSS 308 strain was used in Run I and the COBB 500 strain was used in Run II.

SELECTION AND PREPARATION OF EYES FOR THE TEST
-Eye selection
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 fluorescein-treated 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.

- Eye examination and acclimatisation 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). Too much pressure on the eye from the clamp was avoided. Due to 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 to 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 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, acclimatisation 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 acclimatisation and treatment periods.

THE BASELINE ASSESSMENTS
At the end of the acclimatisation 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. Slight changes in thickness (0.0 to -1.6 %) were observed in the eyes; this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

Vehicle:

physiological saline

Controls:

other: Corneas were exposed to both positive (30 mg of powdered Imidazole) and negative (physiological saline) concurrent controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg; the test material was applied as supplied (after being further powdered).

Duration of treatment / exposure:

10 seconds from the end of the application to the cornea surface

Observation period (in vivo):

4 hours

Number of animals or in vitro replicates:

3 corneas were exposed to the test material.

Details on study design:

TEST PROCEDURE
-Treatment
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test material was applied onto the centre of the cornea, taking care not to damage or touch the cornea. The positive control eyes were treated in a similar way with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline. In Run 1 and Run 2 one eye was treated with physiological saline, three eyes with test material and another three eyes with powdered Imidazole

- Test material removal
Washing (if done): After 10 seconds the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed.
Time after start of exposure: 10 seconds from the end of the application the cornea surface

OBSERVATIONS
-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. A Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Irritation parameter:

other: ICE Class

Basis:

mean

Remarks on result:

other: The ICE class was I

Irritant / corrosive response data:

In the first run, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by a second experiment (Run 2). The second run confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test material is not an irritant.
The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Table 1: Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change in Run 1

	Test Material		Positive Control		Negative Control
Observation	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min (%)	0.0	I	13.6	III	0.0	I
Mean maximum corneal swelling at up to 240 min (%)	0.0	I	34.3	IV	0.0	I
Mean maximum corneal opacity	0.5	I	4.00	IV	0.00	I
Mean fluorescein retention	0.33	I	3.00	IV	0.00	I
Other Observations	The test material 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.		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.		None
Overall ICE Class	3 x I		3 x IV		3 x I

Table 2: Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change in Run 2

	Test Material		Positive Control		Negative Control
Observation	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min (%)	0.6	I	14.0	III	1.6	I
Mean maximum corneal swelling at up to 240 min (%)	2.2	I	32.9	IV	1.6	I
Mean maximum corneal opacity	0.17	I	4.00	IV	0.00	I
Mean fluorescein retention	0.0	I	3.00	IV	0.00	I
Other Observations	The test material 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.		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.		None
Overall ICE Class	3 x I		3 x IV		3 x I

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test material was found to be non irritating to the eye.

Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions

The in vitro eye irritation study was performed in isolated chicken’s eyes. Three eyes were treated with 30 mg of the test material (further powdered). The three positive control eyes were treated in a similar way with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % NaCl solution).

After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

In the first run, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by a second experiment (Run 2). The second run confirmed the negative results.

The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Under the conditions of this study the test material was found to be non 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/Irritation

A study was performed in vitro to assess the irritancy potential of the test material 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 in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Disks of EPISKIN (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material 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 (five replicates) and positive controls (three replicates), respectively.

An additional disk was used to provide an estimate of colour contribution from the test material. Furthermore, three additional test material treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test material. 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 material is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 85.9 % compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin under the conditions of this study. The experiment met the validity criteria, therefore the study was considered to be valid.

Eye Irritation

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 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 in vitro eye irritation study was performed in isolated chicken’s eyes. Three eyes were treated with 30 mg of the test material (further powdered). The three positive control eyes were treated in a similar way with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % NaCl solution).

After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

In the first run, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by a second experiment (Run 2). The second run confirmed the negative results.

The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Under the conditions of this study the test material was found to be non irritating to the eye.

Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one study available.

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 irritation or corrosion.