3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Endpoint summary

Description of key information

In vitro Skin Irritation / Corrosion: Orosz (2018)

The test material is non-irritant to skin.

 

In vitro Eye Irritation: Orosz (2018)

The test material is a non-irritant to the eye.

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:

16 May 2018 - 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH)).

The test material was applied as supplied (although the test material was ground into a powder).

Test system:

human skin model

Remarks:

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

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM) Kit
- Source: SkinEthic, France
- Tissue lot number: 18-EKIN-020
- Expiry date: 21 May 2018

TEST FOR DIRECT MTT REDUCTION
- 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 material 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 should be used to detect and correct for test material interference with the viability measurement.
- Check-method for possible direct MTT reduction with test material: 10 mg of powdered 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.
- If the MTT solution containing the test material turns blue/purple, the test material is presumed to have reduced the MTT.
- After three hours of incubation, yellow colour of the mixture was detected in the test tube. The test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- 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 (e.g. acidified isopropanol) (simulating a tissue humid environment). As the test material had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
- Therefore, in addition to the normal procedure, two additional test material-treated living 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 reading was conducted following the same conditions as for the other tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature - 22.0 - 24.3 °C
- Temperature of post-treatment incubation: 37 °C

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 AND RINSING (Day 0)
- Test material: First an appropriate amount (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 powdered test material 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. The amount was sufficient to cover the epidermal surface.
- Negative and positive controls: 50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette.
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (22.0-24.3°C).
- After the 15 minutes 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 (+ 10 minutes) at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere.

MTT TEST (Day 2)
After 42 hours incubation, all EPISKIN™ (SM) units (except the two living 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 EPISKIN™ (SM) units were incubated for 3 hours 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 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.

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 (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.

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

DATA EVALUATION
- The test material is considered to be corrosive to skin if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is less or equal (≤) to 50 % of the mean viability of the negative controls (Category 2 or Category 1).
- The test material is considered to be non-corrosive to skin if if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is more than (˃) to 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

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):

Incubated at 37 °C for 42 hours followed by 3 hours with MTT

Number of replicates:

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

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Run / experiment:

Test material

Value:

97.4

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:

ADDITIONAL CONTROLS
- As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- As the test material was coloured (off-white), two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.011, Non Specific Colour % was calculated as 1.4 %. This value was below 5%, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The mean OD values for the test material treated skin samples showed 97.4% relative viability compared to the negative control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.753). Standard deviation of the viability results for negative control samples was 2.0 %.
- Acceptance criteria met for positive control: The positive control treated tissues showed 9.7 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.6 %.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 3.2 %.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

HISTORICAL CONTROL DATA

	Negative control (PBS)	Positive control (5 % (w/v) SDS solution)
Mean optical density (OD)	0.788	0.065
Standard deviation	0.129	0.041
Minimum optical density (OD)	0.573	0.019
Maximum optical density (OD)	1.362	0.354
Number of cases	251	246

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

OD: Optical density (absorbance)

Note: All OD values (measured at 570 ± 30 nm) are background corrected values.

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

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

Executive summary:

An in vitro skin irritation study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions using the EPISKIN™ reconstructed human epidermis model.

During the study, disks of EPISKIN™ (SM) (three units) were treated with the powdered 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 disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere, 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 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 97.4 % 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:

2 May 2018 - 22 May 2018

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)

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH))
Test material solubility: The solubility of the test material was tested in physiological saline prior to the experiment (30 mg test material in 1 mL physiological saline). The test material did not dissolve.

The test material was applied in its original form (although it was ground to a fine powder).

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Characteristics of donor animals: approximately 7 weeks old
- Chicken heads were collected after slaughter in a commercial abattoir from chickens.
- Storage, temperature and transport conditions of ocular tissue: 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:
- 30 mg of powdered test material

Negative control eye:
- 30 μL of physiological saline

Positive control eyes:
-30 mg powdered Imidazole.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

One eye was treated with the negative control (physiological saline), three eyes with the test material and another three with the positive control (powdered Imidazole) in each experiment. There were 2 experiments in total.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED 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.
- 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.

EQUILIBRATION AND BASELINE RECORDINGS
- 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 avoiding 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.
- 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, 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 acclimatization and treatment periods.
- 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. 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.

TREATMENT
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 powdered test material 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.

NUMBER OF REPLICATES
- Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

NEGATIVE CONTROL USED
- 30 µL of physiological saline

POSITIVE CONTROL USED
- 30 mg of powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME
- 30 mg of test material for 10 seconds

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.

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 at each time point of both experiments when the test material or positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- 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.

CORNEAL SWELLING CALCULATION

CS at time t = [(CT at time t – CT at time=0)/ CT at t=0] x 100

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 CALCULATION

ΔCO at time t = CO at time t – CO at time 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 time 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 240 min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

FLUORESCEIN RETENTION CALCULATION

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

ICE CLASSIFICATION CRITERIA

- Corneal Thickness:
Mean Corneal Swelling 0 to 5 %: ICE Class I
Mean Corneal Swelling >5 to 12 %: ICE Class II
Mean Corneal Swelling >12 to 18 % ( >75 min after treatment ): ICE Class II
Mean Corneal Swelling >12 to 18 %( ≤75 min after treatment ): ICE Class III
Mean Corneal Swelling >18 to 26 %: ICE Class III
Mean Corneal Swelling >26 to 32 % ( >75 min after treatment): ICE Class III
Mean Corneal Swelling >26 to 32 % ( ≤75 min after treatment ): ICE Class IV
Mean Corneal Swelling >32 %: ICE Class IV

- Corneal Opacity:
Mean Maximum Opacity Score 0.0 - 0.5: ICE Class I
Mean Maximum Opacity Score 0.6 - 1.5: ICE Class II
Mean Maximum Opacity Score 1.6 - 2.5: ICE Class III
Mean Maximum Opacity Score 2.6 – 4.0: ICE Class IV

- Fluorescein Retention (score at 30 minutes post-treatment).
Mean Fluorescein Retention 0.0 - 0.5: ICE Class I
Mean Fluorescein Retention 0.6 - 1.5: ICE Class II
Mean Fluorescein Retention 1.6 - 2.5: ICE Class III
Mean Fluorescein Retention 2.6 – 3.0: ICE Class IV

CLASSIFICATION
- In the case where the result indicates Non-irritant or Corrosive/Severely Irritating, then the test material can be classified. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.

- No category: 3 ×I or 2×I, 1×II

- No prediction can be made: Other combinations

- Category 1: 3×IV or 2×IV, 1×III or 2×IV, 1×II or 2×IV, 1×I,
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of epithelium (in at least 1 eye).

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 - 75 minutes

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 - 240 minutes

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 1

Value:

0.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 2 - 75 minutes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 2 - 240 minutes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 2

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 2

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

Experiment 1
- The test material was stuck on all cornea surfaces after the post-treatment rinse. Two (2/3) cornea surfaces were cleared at 180 minutes after the post-treatment rinse. All cornea surfaces (3/3) were cleared at 240 minutes after the post-treatment rinse.
- Overall ICE Class: 2xI 1xII

Experiment 2
- The test material was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.
- Overall ICE Class: 3xI

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. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes with the test material, the test material was a non-irritant.

POSITIVE CONTROL
- The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

NEGATIVE CONTROL
- The negative control (Physiological saline) was classified as non-irritating, UN GHS Classification: No Category.

TEST 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 each experiment. This study was considered to be valid.

Experiment I Results

Observation	Test Material		Positive Control		Negative Control
	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.5 %	I	11.4 %	II	0.0 %	I
Mean maximum corneal swelling at up to 240 min	0.5 %	I	26.5 %	III	0.0 %	I
Mean maximum corneal opacity	0.50	I	4.00	IV	0.00	I
Mean fluorescein retention	0.83	II	3.00	IV	0.00	I
Other Observations	Test material was stuck on all cornea surfaces after the post-treatment rinse. Two (2/3) cornea surfaces were cleared at 180 minutes after the post-treatment rinse. All cornea surfaces (3/3) were cleared at 240 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	2xI 1xII		1xIII 2xIV		3xI

 

Experiment II Results

Observation	Test Material		Positive Control		Negative Control
	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0 %	I	9.6 %	II	0.0 %	I
Mean maximum corneal swelling at up to 240 min	0.0 %	I	26.1 %	III	0.0 %	I
Mean maximum corneal opacity	0.50	I	4.00	IV	0.00	I
Mean fluorescein retention	0.17	I	3.00	IV	0.00	I
Other Observations	Test material was stuck on all cornea surfaces after the post-treatment rinse. All 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	3xI		1xIII 2xIV		3xI

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

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

Executive summary:

An in vitro eye irritation study was performed in accordance with the standardised guidelines OECD 438, under GLP conditions using isolated chicken's eyes. 

In each experiment after the zero reference measurements, the eye was held in horizontal position and powdered 30 mg test material 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 study was considered to be valid.

More specifically, in Experiment I, no significant corneal swelling was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change and slight fluorescein retention change was observed on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. Two (2/3) cornea surfaces were cleared at 180 minutes after the post-treatment rinse. All cornea surfaces (3/3) were cleared at 240 minutes after the post-treatment rinse.

In Experiment II, no corneal swelling was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change was observed on all three eyes. No significant fluorescein retention change was noted on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. The all cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.

Therefore, under the conditions of the study, the test material is a non-irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro Skin Irritation / Corrosion: Orosz (2018)

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

During the study, disks of EPISKIN™ (SM) (three units) were treated with the powdered 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 disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere, 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 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 97.4 % 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.

 

In vitro Eye Irritation: Orosz (2018)

An in vitro eye irritation study was performed in accordance with the standardised guidelines OECD 438, under GLP conditions using isolated chicken's eyes. 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 powdered 30 mg test material 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 study was considered to be valid.

More specifically, in Experiment I, no significant corneal swelling was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change and slight fluorescein retention change was observed on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. Two (2/3) cornea surfaces were cleared at 180 minutes after the post-treatment rinse. All cornea surfaces (3/3) were cleared at 240 minutes after the post-treatment rinse.

In Experiment II, no corneal swelling was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change was observed on all three eyes. No significant fluorescein retention change was noted on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. The all cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.

Therefore, under the conditions of the study, the test material is a non-irritant to the eye.

 

Justification for classification or non-classification

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