C.I. Reactive Yellow 2

Endpoint summary

Description of key information

Skin Irritation: In Vitro

Under the conditions of the study, the test material is non-irritant to skin.

Eye Irritation: In Vitro

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

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August 2016 to 12 August 2016

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

Remarks:

EPISKIN™ (SM) reconstituted human epidermis (three-dimensional human epidermis model)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

SOURCE ANIMAL
Not applicable; EPISKIN™ (SM) reconstituted human epidermis (three-dimensional human epidermis model).

Justification for test system used:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 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.
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). The kits were found to be in good order.
The EPISKIN™ (SM) kit was kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.
Procedures described were performed under aseptic conditions (in sterile hood using sterile equipment).

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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (24.4 – 26.4 °C).
- Temperature of post-treatment incubation: 37 °C
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. If the test item was stuck on the surface of the epidermis additional rinsing was used. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: None specified
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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: After the 42 hours incubation, all EPISKIN™ (SM) units (except of the two living colour control units) were transferred into the MTT working solution filled with 2 mL of 0.3 mg/mL MTT per well.
- Incubation time: 3 hours (± 5 mins) at 37 °C with 5 % CO2 protected from light.
- Wavelength: 570 nm
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 09 August 2016) was stored in refrigerator (2-8 °C) protected from light. It was diluted with pre-warmed (37 °C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

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 (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.

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.

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

NUMBER OF REPLICATE TISSUES: In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test items were coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls are used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are as follows:
Approximately 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: Yellow
-Test items reacting with MTT: Blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.
Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test items had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.

PREDICTION MODEL / DECISION CRITERIA
The test item is considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg of the test item was applied evenly to the epidermal surface. The amount was sufficient to cover the epidermal surface.

NEGATIVE CONTROL
- Amount applied: 50 μL of negative control (Phosphate Buffered Saline (PBS)) was added to each skin unit by using a suitable pipette. The negative control was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

POSITIVE CONTROL
- Amount(s) applied: The positive control 5 % (w/v) Sodium Dodecyl Sulphate solution was added to each skin unit by using a suitable pipette. The positive control was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- Preparation: The positive control solution was prepared freshly in the testing laboratory.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2.

Number of replicates:

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

Irritation / corrosion parameter:

other: Relative viability percentage

Run / experiment:

Mean

Value:

96.9

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:

OTHER EFFECTS:
- Visible damage on test system: None specified
- Direct-MTT reduction: As no colour change (yellow colour) was observed after three hours of incubation of the test items in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Colour interference: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.015, Non Specific Colour % was calculated as 2.0 %. This value was below 5 %, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
The OD values for the test item treated skin samples showed 96.9 % relative viability. This is above the threshold of 50 % and therefore the test item was considered non-irritant to the skin.
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.748). Standard deviation of the viability results for negative control samples was 9.1.
- Acceptance criteria met for positive control: The positive control treated tissues showed 8.8 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 4.2.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 10.2. The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test material is non-irritant to 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.

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

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

Following exposure with the test material, the mean cell viability was 96.9 % compared to the negative control. This is above the threshold of 50 %, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of the study the test material is non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August 2016 to 29 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

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: COBB 500 and ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and heads transported to the testing laboratory 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).
- Time interval prior to initiating testing: The heads were received at the test site and processed within 2 hours and 15 minutes of collection.
- indication of any existing defects or lesions in ocular tissue samples: 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item.

Duration of treatment / exposure:

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

Observation period (in vivo):

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.

Duration of post- treatment incubation (in vitro):

240 minutes (4 hours)

Number of animals or in vitro replicates:

3 corneas were exposed to the test material.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED 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.
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

EQUILIBRATION AND BASELINE RECORDINGS
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatization and treatment periods.
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

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

NEGATIVE CONTROL USED
In each experiment negative control eye was treated with 30 μL of physiological saline.

POSITIVE CONTROL USED
Positive control eyes were treated with 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 item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
An exposure period of 10 seconds from the end of the application was used.

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 Bern 900 slit-lamp microscope was used for the measurements.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 2x20 mL saline after treatment in each experiment.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated according to the following formulae:
ΔCO at time t = CO at time t – CO at t = 0
Mean ΔCOmax = [FECOmax(30 to 240 min) + SECOmax(30 to 240 min) + TECOmax(30 to 240 min)] / 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 time t and corneal opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO = third eye cornea opacity
max(30 to 240 min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline corneal opacity of the individual eye

- Damage to epithelium based on fluorescein retention:
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 time t 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

- Swelling:
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).

Irritation parameter:

other: ICE Class

Run / experiment:

Mean

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Acceptance criteria met for negative control: The positive control (Imidazole) was determined to be severely irritating. Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse. Overall ICE Class: 1 x III, 2 x IV in both experiments.

- Acceptance criteria met for positive control: The negative control physiological saline was determined to be non-irritating. Overall ICE Class: 3 x I in both experiments.

- Range of historical values if different from the ones specified in the test guideline: The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Irritant / corrosive response data:

The test material showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test material was non-irritant.
In both experiments test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse. Overall ICE Class: 2 x I, 1 x II in both experiments.

Validity

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

Interpretation of results:

GHS criteria not met

Conclusions:

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

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.

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 the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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

For Experiment 1, no significant corneal swelling (mean ≤5 %) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

For Experiment 2, no significant corneal swelling (mean ≤5 %) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Under the conditions of the test, the test item was determined to be a non-irritant based on the in vitro eye irritation assays in isolated chicken eyes.

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

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 assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

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

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

Following exposure with the test material, the mean cell viability was 96.9 % compared to the negative control. This is above the threshold of 50 %, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of the study the test material is non-irritant to skin.

Eye Irritation: In Vitro

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 assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

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 the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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

For Experiment 1, no significant corneal swelling (mean ≤5 %) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

For Experiment 2, no significant corneal swelling (mean ≤5 %) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Under the conditions of the test, the test item was determined to be a non-irritant based on the in vitro eye irritation assays in isolated chicken eyes.

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 irritation and eye irritation.