2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Endpoint summary

Description of key information

Skin Irritation (in vitro)

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

Eye Irritation (in vitro)

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

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:

24 May 2017 to 26 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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
> Living Tissues
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic, France.
- Tissue lot number: 17-EKIN-021
- Expiry date: 29 May 2017

> Killed Tissues
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic, France.
- Tissue lot number: 17-EKIN-003
- Expiry date: 23 January 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (24.9 - 26.8°C)
- Temperature of post-treatment incubation: 37°C

ASSESSMENT OF POSSIBLE MTT REDUCTION
10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded.
After three hours incubation, purple red colour was detected in the test tube. Thus, the test material reacted with MTT and therefore the use of additional controls was necessary.

ASSESSMENT OF COLOURING POTENTIAL OF TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or extracting solution. The test item was shown being an MTT-interacting substance and the test material had an intrinsic colour a third set of controls was necessary. To avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled) was needed.

Two additional test item-treated living tissues and two additional test item-treated killed tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

MAIN STUDY

PRE-INCUBATION (DAY -1)
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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 OF TEST MATERIAL (DAY 0)
10 μL distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test item was applied evenly to the epidermal
surface. The test material was spread gently on the skin surface with a pipette tip without damaging the epidermis.
50 μL of negative control or positive control were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly without damaging the epidermis.

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period, each tissue was removed from the well and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface. 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 (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

MTT TEST (DAY 2)
After the 42 hours incubation, all tissues (except of two NSClving and two NSCkilled colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred tissues were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

FORMAZAN EXTRACTION (DAY 2)
After the incubation with MTT, a formazan extraction was undertaken. A disc 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 (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.

NUMBER OF REPLICATE TISSUES: 3 (normal procedure) plus an additional two test material treated killed epidermis and two negative control treated killed epidermis was used in the study

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate at the required wavelength on each day before use.



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:
- Relative mean tissue viability is ≤ 50%: Irritant (H315 Category 2)
- Relative mean tissue viability is > 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:
- Positive Control: 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%.
- Negative Control: 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%.
- Test Material: 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 mg wetted with 10 µL distilled water to improve contact

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 (normal procedure) plus an additional two test material treated killed epidermis and two negative control treated killed epidermis was used in the study

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

63.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

As the test material was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissue was 0.014, Non-Specific Colour % was calculated as 2.0% (see Table 1). This value was below 5%, therefore additional data calculation was not necessary.

As colour change (purple red) 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 necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in Table 2. Based on the observed mean OD (-0.009), the calculated NSMTT% is -1.3%. Since the OD values were negative, therefore these values excluded from the NSMTT calculation.

As the test material was shown being an MTT-interacting substance and the test material had an intrinsic colour, two additional test material-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.021, Non-Specific Colour % (NSCkilled%) was calculated as 3.1% (see Table 3). Because the NSCliving% was not used, correction with NSCkilled% was not necessary.

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 4. The OD values for the test material treated skin samples showed 63.1% relative viability.

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 of the test material was performed in a reconstructed human epidermis model. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 439 and EU Method B.46.

During the study discs of EPISKIN^TM (SM) (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, in a >95% humidified atmosphere. The viability of each disc was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control).

Two additional discs were used to provide an estimate of colour contribution (NSCliving) from the test material. The possible MTT interaction potential of the test material was examined using two additional test item treated killed epidermis units and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with test material, the mean cell viability was 63.1% compared to the negative control. This is above the threshold of 50%, therefore the test

material was considered as being non-irritant to skin. 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:

18 May 2017 to 16 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500 (Experiment I) and ROSS 308 (Experiment II)

Details on test animals or tissues and environmental conditions:

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

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Number of animals or in vitro replicates:

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

Details on study design:

SELECTION OF EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the 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.

BASELINE RECORDINGS
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. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

EXAMINATION OF EYES 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). Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, 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.


NUMBER OF REPLICATES
One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

NEGATIVE CONTROL USED
30 μL of physiological saline

POSITIVE CONTROL USED
30 mg powdered imidazole

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

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

REMOVAL OF TEST SUBSTANCE
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 20 mL saline was performed after treatment and at each time point when the test item or positive control material remaining on the cornea was observed in each experiment. The test material treated eyes were rinsed additional gentle rinsing with 3x20 mL saline after treatment in each experiment.

EVALUATION
Corneal swelling was calculated according to the following formulae:

CS at time t = [(CT at time t –CT at t=0) / CT at t=0] x100

Mean CS at time t = [FECS(at time t)+ SECS(at time t) + TECS(at time t)] / 3

where
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point

Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = [FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3

where
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t – FR at t=0

Mean ΔFR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3

where
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Run / experiment:

Experiment I

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

Experiment I

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Run / experiment:

Experiment II

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

Experiment II

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

The test material showed no significant corneal effect in the first experiment. As the test material was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (the negative control physiological saline was classified as non-irritating)
- Acceptance criteria met for positive control: yes (the positive control (imidazole) was classified as severely irritating)
The results from all eyes used met the quality control standards.
- Range of historical values if different from the ones specified in the test guideline: The negative control and positive control results were within the historical control data range in each experiment. This experiment was considered to be valid.

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of the study, the test material was determined to be a non-irritant.

Executive summary:

An in vitro eye irritation study of the test material was performed in isolated chicken's eyes. The irritation effects of the test material were investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 438.

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

treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity

change (severity 0.5) was observed on one eye. No significant fluorescein retention change (severity 0.5) was noted on three eyes. The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse. No other corneal effect was observed.

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

Based on these in vitro eye irritation tests in isolated chicken eyes, the test material was determined to be a non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation (in vitro)

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

During the study discs ofEPISKIN^TM(SM) (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, in a >95% humidified atmosphere. The viability of each disc was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control).

Two additional discs were used to provide an estimate of colour contribution (NSCliving) from the test material. The possible MTT interaction potential of the test material was examined using two additional test item treated killed epidermis units and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with test material, the mean cell viability was 63.1% compared to the negative control. This is above the threshold of 50%, therefore the test

material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Eye Irritation (in vitro)

An in vitro eye irritation study of the test material was performed in isolated chicken's eyes. The irritation effects of the test material were investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 438. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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

treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity

change (severity 0.5) was observed on one eye. No significant fluorescein retention change (severity 0.5) was noted on three eyes. The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse. No other corneal effect was observed.

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

Based on these in vitro eye irritation tests in isolated chicken eyes, the test material was determined to be a non-irritant.

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 corrosion/ irritation or eye irritation.