Condensation products of phenol and salicylaldehyde

Endpoint summary

Description of key information

In vitro Skin Irritation

Under the conditions of this study, the test material is irritating to the skin.

In vitro Eye Irritation

Under the conditions of this study the test material is irritating 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:

23 March 2016 to 25 March 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)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- The test material was applied in its original form, no formulation was required (although the test item was ground to a fine powder).

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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)
- Tissue batch number(s): 16-EKIN-012 and 15-EKIN-045 (for killed epidermis)
- Expiry date: 28 March 2016 and 16 November 2015 (for killed epidermis)
- 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.

KIT RECEPTION
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Check-method for possible direct MTT reduction with test material: Approximately 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: test materials which do not react with MTT: yellow and test materials reacting with MTT: blue or purple. After three hours incubation, purplish colour of the mixture was detected in the test tube. Thus, the test material reacted with MTT and therefore the use of additional controls was necessary.
- Check-method to detect the colouring potential of test-materials: Prior to treatment, the test material was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test materials 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 materials to stain the epidermis by using additional control tissues.
The test material was showed being an MTT-interacting substance and the test material had an intrinsic colour so a third set of controls were necessary. The test material may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled) was needed.
- Therefore, in addition to the normal procedure, two additional test material-treated living tissues and two additional test material-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.
- Process for test materials with both a direct MTT reduction with test material, and dyes and chemicals able to colour the tissue (and possibly affecting the absorption at the measurement wavelength): The test material was identified as producing both direct MTT reduction and colour interference was also require a third set of controls. This is usually the case with darkly coloured test chemicals interfering with the MTT assay (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT. These test chemicals may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of control for non-specific colour in killed tissues (NSCkilled) was performed.

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 (DAY 0)
- Test Material: As the test material was solid, first an appropriate amount (10 μL) of distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material 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.
- 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. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.9 - 26.7 °C).
- As the test material was showed being an MTT-interacting substance, in addition to the normal procedure, two test material treated killed epidermis and two negative control treated killed epidermis was used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.9 - 26.7 °C)
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- 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 test material stuck on 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). 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 TEST AND FORMAZAN EXTRACTION (DAY 2)
- After the 42 hours incubation, all EPISKIN™ (SM) units (except of two 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 (± 5 min) at 37 °C in an incubator with 5 % CO2 protected from light.
- 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.

NUMBER OF REPLICATE TISSUES: 3

VALIDITY OF THE TEST
- The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40 % and the standard deviation value (SD) of the % viability values should be ≤ 18.
- The SD calculated from individual % tissue viability values of the three test material treated replicates should be <18.
- The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

INTERPRETATION OF TEST RESULTS
- The irritation potential of test materials can be classified according to the United Nations Globally Harmonised System of Classification and Labelling of Chemicals [10,11], and a similar system is used in CLP. In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test material. The test material 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. The prediction model (PM) is described below:
Mean tissue viability % is ≤ 50 % = Category 2
Mean tissue viability % is > 50 % = No Category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

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

Duration of treatment / exposure:

15 (± 0.5) minutes

Duration of post-treatment incubation (if applicable):

42 (± 1) hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

14.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ADDITIONAL CONTROLS
- As the test material was coloured, two additional test material-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.049, Non Specific Colour % was calculated as 6.9 %. This value was above 5 %, therefore additional data calculation was necessary.
- As colour change (purplish) was observed after three hours of incubation of the test material in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on these observed mean OD (0.054), the calculated NSMTT is 7.5 %. This was considered to be significant, thus additional data calculation was necessary.
- As the test material was showed 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 0.067, Non Specific Colour % (NSCkilled%) was calculated as 9.5 %. Because the NSCliving% was used, therefore correction with NSCkilled% was necessary.

VIABILITY RESULTS
- The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The OD values for the test material treated skin samples showed 14.9 % relative viability.

VALIDITY OF THE TEST
- After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the three negative control tissues was in the recommended range (0.710). Standard deviation of the viability results for negative control samples was 6.3.
- The positive control treated tissues showed 9.0 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 3.5.
- The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 1.3.
- 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.

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

Treatment	Optical Density (OD)			TODTT	Viability (% RV)
		Measured	Blank Corrected
Negative Control (PBS)	1	0.755	0.708	-	99.7
	2	0.713	0.667	-	93.9
	3	0.802	0.756	-	106.4
	Mean	-	0.710	-	100.0
Positive Control (5 % (w/v) SDS Solution)	1	0.095	0.049	-	6.9
	2	0.139	0.093	-	13.0
	3	0.098	0.051	-	7.2
	Mean	-	0.064	-	9.0
Test Material	1	0.187	0.140	0.105	14.8
	2	0.197	0.150	0.115	16.2
	3	0.179	0.132	0.097	13.6
	Mean		0.141	0.106	14.9

Mean blank value was 0.046.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

TODTT: The measured values were corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.054) and for non-specific colour reduction (by subtracting the NSCliving value of 0.049 and by added the NSCkilled value of 0.067).

Interpretation of results:

other: EU Criteria: Category 2, Causes skin irritation (H315)

Conclusions:

Under the conditions of this study, the test material is irritating to the skin.

Executive summary:

An in vitro skin irritation test of the test material was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test material was evaluated according to the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

Disks of EPISKIN™ (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. 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 material. The possible MTT interaction potential of the test material was examined using two additional test material treated 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 material is considered to be irritant to skin.

Following exposure to the test material, the mean cell viability was 14.9 % (after adjustment for non-specific MTT reduction, NSCliving and NSCkilled) compared to the negative control. This is below the threshold of 50 %, therefore the test material was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 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)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- The test material was applied as supplied, no formulation was required. Although the test material was further powdered.

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 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). The heads were received at the testing laboratory and processed within approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

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

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Selection: 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: 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 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.
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 acclimatisation and treatment periods.
- At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. Slight corneal thickness changes (0.0 – 1.6 %) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.
- 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 item was applied onto the centre of 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 (Salsol solution, 0.9 % (w/v) NaCl)

POSITIVE CONTROL USED
- 30 mg of powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME
- 30 mg for 10 seconds

REMOVAL OF TEST SUBSTANCE
- 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 when the control material remaining on the cornea was observed.

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.

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.

SCORING SYSTEM:
- Corneal swelling was calculated according to the following formulae:

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 was calculated according to the following formulae:

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 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 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 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

ICE CLASSIFICATION
- 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:
Mean Fluorescein Retention Score at 30 minutes post – treatment 0.0 - 0.5: ICE Class I
Mean Fluorescein Retention Score at 30 minutes post – treatment 0.6 - 1.5: ICE Class II
Mean Fluorescein Retention Score at 30 minutes post – treatment 1.6 - 2.5: ICE Class III
Mean Fluorescein Retention Score at 30 minutes post – treatment 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
- 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).
- No prediction can be made: Other combinations

Irritation parameter:

percent corneal swelling

Run / experiment:

75 minutes

Value:

14.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III

Irritation parameter:

percent corneal swelling

Run / experiment:

240 minutes

Value:

16.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0

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:

TEST MATERIAL
- Results can be seen in Table 1.
- The overall ICE class was 1xI; 1xII; 1xIV, based on these results the test material is classified as severely irritating. UN GHS Classification: Category 1.

POSITIVE CONTROL
- The positive control (Imidazole) was classified as severely irritating.

NEGATIVE CONTROL
- The negative control Physiological saline was non-irritating.

VALIDITY OF THE TEST
- 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.

Table 1: Summary of 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	14.9 %	III	9.5 %	II	0.0 %	I
Mean maximum corneal swelling at up to 240 min	16.5 %	II	22.7 %	III	0.0 %	I
Mean maximum corneal opacity	4.00	IV	4.00	IV	0.00	I
Mean fluorescein retention	0.0	I	3.00	IV	0.00	I
Other Observations	Test material 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.		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	1xI; 1xII; 1xIV		1xIII; 2xIV		3xI

 

Interpretation of results:

other: EU Criteria: Category 1. Causes severe eye damage

Conclusions:

Under the conditions of this study the test material is irritating to the eye.

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 evaluated according to 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 powdered 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 the study, 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 experiment. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. Severe corneal opacity change (severity 4) was observed in all three eyes. No fluorescein retention change was noted on all three eyes.

The test material was fully stuck to the cornea and could not be washed off during the study. The assessment was based on the examination of the free corneal surfaces.

Under the conditions of this study the test material is irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

In vitro Skin Irritation

An in vitro skin irritation test of the test material was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test material was evaluated according to the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Disks of EPISKIN™ (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. 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 material. The possible MTT interaction potential of the test material was examined using two additional test material treated 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 material is considered to be irritant to skin.

Following exposure to the test material, the mean cell viability was 14.9 % (after adjustment for non-specific MTT reduction, NSCliving and NSCkilled) compared to the negative control. This is below the threshold of 50 %, therefore the test material was considered as being irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is irritating to the skin.

In vitro Eye Irritation

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 evaluated according to the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After the zero reference measurements, the eye was held in horizontal position and 30 mg powdered 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 the study, 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 experiment. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. Severe corneal opacity change (severity 4) was observed in all three eyes. No fluorescein retention change was noted on all three eyes.

The test material was fully stuck to the cornea and could not be washed off during the study. The assessment was based on the examination of the free corneal surfaces.

Under the conditions of this study the test material is irritating 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 is classified as Category 2: Causes skin irritation (H315) and Category 1: Causes serious eye damage (H318).