2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Endpoint summary

Description of key information

Not skin irritating

Able to cause serious eye irritation (Eye Irrit. 2 (H319), according to the CLP Regulation (EC) 1272/2008)

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:

weight of evidence

Study period:

February from 01 to 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was applied in its original form, no formulation was required.

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and non-skin irritating test substances.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model, three-dimensional human epidermis model.
- Source: EPISKIN SNC Lyon, France.
- Tissue batch number: 17-EKIN-005
- Seeding: adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Culturing: a highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Demonstration of proficiency: prior to routine use of the method, the testing laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.
- Health check: all biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

CONDITIONS USED FOR TEST SYSTEM
- Conditions used during treatment / exposure: 37 °C in an incubator with 5 % CO2, ≥ 95 % humidified atmosphere.
- Conditions of post-treatment incubation: 37 °C in an incubator with 5 % CO2, ≥ 95 % humidified atmosphere.

PRE-INCUBATION, day [-1]-0
- Filling volume: the appropriate number of an assay plate wells were filled with the pre-warmed medium, 2 ml per well.
- Incubation: the epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight, 18 - 24hours.

APPLICATION, day 0
- Pre-treatment: the epidermal surface was first moistened with 5 µl deionised water in order to improve further contact between powder and epidermis.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing thoroughly with approximately 25 ml PBS 1x solution. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

POST-INCUBATION, day 0-2
- Incubation after rinsing: units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 ml/well).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT stock solution: dissolved to a final concentration of 3 mg/ml in saline buffer. The obtained stock solution can be stored in refrigerator (2-8 °C), protected from light up to 15 days.
- MTT concentration: approximately 10 mg test item was added to 2 mL MTT 0.3 mg/ml solution and mixed.
- Incubation time: incubation for three hours at 37 °C protected from light.
- Plate: the epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µl acidified isopropanol (one tube corresponding to one well of the tissue culture plate). The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol.
- Duration of incubation: approximately four hours.
- Conditions of incubation: room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY, day 2
Following the formazan extraction, 2×200 µl sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm (± 10 nm; Read out range: 0-3.5 Abs) using acidified isopropanol solution as the blank (6×200 µl).

ACCEPTANCE CRITERIA
- 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 should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
- for test chemicals, the standard deviation value (SD) of the % viability between tissue replicates should be ≤ 18.

PREDICTION MODEL / DECISION CRITERIA
- The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), 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 substance may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg, applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

NEGATIVE CONTROL
- Amount applied: 10 µl applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

POSITIVE CONTROL
- Amount applied: 10 µl applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

Test item: 3 replicates
Positive and negative controls: 3 replicates
Colour control: 2 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

106

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The OD values obtained for the three replicates of the test item were 0.752, 0.979 and 0.755, corresponds to 96, 125 and 96 % viability, when compared to the results obtained from the negative control.

ACCEPTANCE CRITERIA
The mean OD value of the three negative control tissues was 0.786.
The mean OD value obtained for the positive control was 0.163 and this result corresponds to 21 % viability when compared to the results obtained from the negative control.
Each calculated standard deviation value (SD) for the % viability was below 18.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Possible direct MTT reduction with test item: no colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item: mean OD (measured at 570 nm) of these tissues was determined as 0.018. The Non Specific Colour % (NSC %) was calculated as 2.3 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

OD values and viability percentages of test item

Substance	Optical Density (OD)		Viability (%)
Test item	1	0.752	96
	2	0.979	125
	3	0.755	96
	mean	0.829	106
	standard deviation (SD)		16.57

OD values and viability percentages of the controls

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.812	103
	2	0.721	92
	3	0.824	105
	mean	0.786	100
	standard deviation (SD)		7.15
Positive Control: SDS (5 % aq.)	1	0.157	20
	2	0.177	23
	3	0.154	20
	mean	0.163	21
	standard deviation (SD)		1.59

OD values and NSC % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour % (NSC %)
Test item treated tissues without MTT incubation	1	0.02	2.3
	2	0.017
	mean	0.018

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

The test item reveals no skin irritation potential under the utilised testing conditions.

Executive summary:

EpiSkinTM SM test has been performed to predict the irritation potential of the test item, by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item has an intrinsic colour, therefore two additional test item treated tissues were used for the non-specific OD evaluation. No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Conclusion

The test item reveals no skin irritation potential under the utilised testing conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 31, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was finely ground before application.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary.
- Characteristics of donor animals: approximately 7 weeks old, 1.5 – 2.5 kg; only the eyes of healthy animals, considered suitable for entry into the human food chain, were used.
- Collection: head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current).
- Health inspection: after collection, the heads were inspected for appropriate quality.
- Storage, temperature and transport conditions of ocular tissue: after collection, heads were wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box). The ambient temperature was optimal (19.4 – 20.3 ºC) during the transport.
- Time interval prior to initiating testing: use approximately within 2 hours from collection.
- Eyes: all eyes used in the assay were from the same groups of eyes collected on one specific day.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied in its original form, no formulation was required.

Controls:

yes

Amount / concentration applied:

The subsatnce was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Each treatment group and concurrent positive control consists of 3 eyes. The negative control group consists of 1 eye.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eye 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 fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 ml isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of Eyes
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. 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.

Eyes Examination and Acclimatization Time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). 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 applied 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes or 0.1 to 0.15 ml/min. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The flow of saline to the eyes by the mechanical pump was monitored.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. 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.

ACCLIMATIZATION
Acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
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 ± 5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NEGATIVE CONTROL USED
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µl from micropipette, in such a way that the entire surface of the cornea was covered with negative control.

POSITIVE CONTROL USED
Three positive control eyes were treated in a similar way as for test item, appling 0.03 g of imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea.

REMOVAL OF TEST SUBSTANCE
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 ml saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

The imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 ml saline was performed in all imidazole and test item treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

OBSERVATIONS
The cornea thickness, cornea opacity and morphological effects (e.g., pitting or loosening of the epithelium) were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

Retention of Corneas: at the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

METHODS FOR MEASURED ENDPOINTS and SCORING SYSTEM

CORNEA SWELLING
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope (slit-width setting: 9½, equal to 0.095 mm).
The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score is then given.
For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5 %) following application are counted as zero. Large negative numbers (> -12 %) 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.

Scoring system - ICE classification criteria for corneal swelling
Mean Corneal Swelling (%) // ICE Class
0 to 5 I
> 5 to 12 II
> 12 to 18 ( >75 min after treatment ) II
> 12 to 18 ( <75 min after treatment ) III
> 18 to 26 III
> 26 to 32 ( >75 min after treatment ) III
> 26 to 32 ( <75 min after treatment ) IV
> 32 IV

The four categories of ICE Class are:
- No swelling I
- Slight swelling II
- Moderate swelling III
- Severe swelling IV

CORNEA OPACITY
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal opacity was evaluated by using the area of the cornea that is most densely opacified for scoring.
Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score is then given.

Scoring system - ICE classification criteria for corneal opacity
Corneal opacity score:
0: no opacity
0.5: very faint opacity
1: scattered or diffuse areas; details of the iris are clearly visible
2: easily discernible translucent area; details of the iris are slightly obscured
3: severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: complete corneal opacity; iris invisible

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

The four categories of ICE Class are:
- No swelling I
- Slight swelling II
- Moderate swelling III
- Severe swelling IV

FLUORESCEIN RETENTION
The fluorescein retention was measured on two occasions, baseline (t=0) and 30 minutes after the post-treatment rinse.
The mean fluorescein retention value of all test eyes was calculated for the 30-minute observation time point, and used for the overall category score.

Scoring system - ICE classification criteria for fluorescein retention
Flurescein retention score:
0: no fluorescein retention
0.5: very minor single cell staining
1: single cell staining scattered throughout the treated area of the cornea
2: focal or confluent dense single cell staining
3: confluent large areas of the cornea retaining fluorescein

Mean fluorescein retention score // ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 3.0 IV

The four categories of ICE Class are:
- No swelling I
- Slight swelling II
- Moderate swelling III
- Severe swelling IV

INTERPRETATION OF RESULTS
Once each endpoint has been evaluated and the ICE classes has been assigned, the in vitro classification for a test chemical is assessed by reading the GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity, and fluorescein retention as described below.

UN GHS Classification, combinations of the 3 Endpoints
- No Category:
3 x I
2 x I,1 x II
- Category 1 (Causes serious eye damage)
3 × IV
2 × IV, 1 × III
2 × IV, 1 ×II (combinations of categories less likely to occur)
2 × IV, 1 × I (combinations of categories less likely to occur)
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 the epithelium (in at least 1 eye)
- No prediction can be made: other combinations

Irritation parameter:

in vitro irritation score

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item did not cause ocular corrosion or severe irritation (overall ICE class: 1xI, 1xII, 1xIV)

Other effects / acceptance of results:

Under tested consitions, test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE score was 1xI, 1xII, 1xIV.

According to the guideline OECD 438, the overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made”.

No pitting of corneal epithelial cells, loosening of epithelium, roughening of the corneal surface and sticking of the test substance to the cornea were noted.

CONTROLS
Positive and negative control values were within the corresponding historical control data ranges.
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
The negative control NaCl (9 g/l saline) had no significant effects on the chicken eye in this study.

Interpretation of results:

other: neither classified as an ocular corrosive nor severe eye irritant, nor not classified.

Conclusions:

The substance overall in vitro classification is neither classified as an ocular corrosive or severe eye irritant, nor not classified.

Executive summary:

The Isolated Chicken Eye Test (ICET) system was used to evaluate the potential ocular corrosivity and irritancy of the test item, by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item and imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study.

One negative control eye was treated with 30 µl saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 ml saline solution at ambient temperature and this procedure was repeated for each eye.

Adherence of the test item and the positive control imidazole was observed on the cornea surfaces at 240 min after the post-treatment rinse.

Under test conditions, the substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 1xII, 1xIV. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Conclusion

According to the guideline OECD 438, the substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made”.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 02 to 07 November, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A single application of 0.1 ml (96 mg) of the test material was administered to the non irrigated eye.

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

Three

Details on study design:

Ocular reactions were recorded 1, 24, 48 and 72 hours,7, 14 and 21 days after administration.

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

1 - 2

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 72 hrs

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

2

Reversibility:

other: in one animal effects have not fully reversed within the observation period of 21 days

Irritation parameter:

chemosis score

Basis:

animal:

Time point:

24/48/72 h

Score:

2 - 2.3

Reversibility:

fully reversible within: 21 days

Irritant / corrosive response data:

The test material produced scattered or diffuse to opalescent corneal opacity, iridial inflammation and severe conjunctival irritation. Vascularisation, with a localised ingrowth of vessels for 2 mm to 3 mm in length, was noted in one treated eye at the 7, 14 and 2l day observations. Orange coloured staining of the cornea and conjunctivae was noted in all treated eyes.
The staining did not affect evaluation of ocular effects. Orange coloured staining of the fur around all treated eyes was noted throughout the study.
The vascularisation noted in one treated eye at the 21-day observation was considered to be indicative of irreversible ocular damage.
The test material was classified as capable to cause serious eye damage.

Individual and Mean Scores for Cornea, Iris and Conjunctivae for EU Labelling Regulations

Rabbit Number and Sex	Time after treatment	Comeal Opacity	Iris	Redness	Chemosis
191 Male	24 Hours	1	1	2	2
	48 Hours	2	1	2	2
	72 Hours	2	1	2	2
Total		5	3	6	6
Mean		1.7	1.0 +	2.0	2.0 +
68 Male	24 Hours	1	1	2	2
	48 Hours	1	1	2	2
	72 Hours	1	1	2	2
Total		3	3	6	6
Mean		1.0	1.0 +	2.0	2.0 +
69 Male	24 Hours	1	1	2	3
	48 Hours	2	1	2	2
	72 Hours	3	1	2	2
Total		6	3	6	7
Mean		2.0 +	1.0 +	2.0	2.3 +

+ : Positive criteria

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	191 Male						191 Male							191 Male
Time After Treatment	1 h	24 h	48 h	72 h	7 d	14 d	1 h	24 h	48 h	72 h	7 d	14 d	21 d	1 h	24 h	48 h	72 h	7 d	14 d
CORNEA
Degree of Opacity	0s	1	2	2	0	0	0s	1s	1	1	1V	1V	0V	0s	1	2	3	0	0
Area of Cornea Involved	0	2	2	2	0	0	0	2	3	3	2	2	0	0	3	2	2	0	0
IRIS	0	1	1	1	0	0	1	1	1	1	0	0	0	1	1	1	1	0	0
CONJUNCTIVAE
Redness	2s	2	2	2	1	0	2s	2s	2	2	2	2	1	2s	2s	2	2	2	0
Chemosis	3	2	2	2	1	0	3	2	2	2	1	1	0	3	3	2	2	1	0
Discharge	3Sf	3Sf	3Sf	2Sf	1Sf	0Sf	3Sf	2Sf	2Sf	2Sf	2Sf	2Sf	1Sf	3Sf	3Sf	3Sf	3Sf	1Sf	0Sf

s: Orange coloured staining

V: Vascularisation, with an ingrowth of vessels for 2 mm to 3 mm in length

Sf : Orange coloured staining of the fur around the treated eye

Interpretation of results:

other: Eye Dam. 1 (H318), according to the CLP Regulation (EC) No 1272/2008

Conclusions:

The test material was classified as capable to cause serious eye damage.

Executive summary:

A study was performed to assess the irritation of the test material to the eye of the New Zealand White rabbit. The method followed OECD Guidelines 405.

A single application of 0.1 ml (96 mg) of the test material was administered to the non irrigated eye of three rabbits. Ocular reactions were recorded 1, 24, 48 and 72 hours,7, 14 and 21 days after administration.

The test material produced scattered or diffuse to opalescent corneal opacity, iridial inflammation and severe conjunctival irritation. Vascularisation, with a localised ingrowth of vessels for 2 mm to 3 mm in length, was noted in one treated eye at the 7, 14 and 21 day observations. Orange coloured staining of the cornea and conjunctivae was noted in all treated eyes. The staining did not affect evaluation of ocular effects. Orange coloured staining of the fur around all treated eyes was noted throughout the study. The vascularisation noted in one treated eye at the 21-day observation was considered to be indicative of irreversible ocular damage. The test material was classified as capable to cause serious eye damage.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were 1 - 2 for corneal opacity, equal to 1 for iritis, equal to 2 for both conjunctival redness and in the range of 2 - 2.3 for conjunctival oedema, in all the three tested animals. Reactions oberved were all reversible within 21 days except in one case: in one animal conjunctival redness effects have not fully reversed within the observation period of 21 days.

Therefore, the substance meets the criteria to be classified as able to cause eye damage, category 1 (i.e. Eye. Dam. 1, H318), according to the CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

SKIN IRRITATION

EpiSkinTM SM test has been performed to predict the irritation potential of the test item, according to the OECD Test Guideline No. 439. The test item has an intrinsic colour, therefore two additional test item treated tissues were used for the non-specific OD evaluation. No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits and the experiment was considered to be valid.

An in vivo study conducted on a less pure substance's lot is also available. The skin of the New Zealand White rabbits was treated. The method followed the OECD Guidelines No. 404. Well-defined erythema was noted at all treated skin sites one hour after patch removal and persisted at the 24-hour observation. Very slight to well-defined erythema was noted at all treated skin sites at the 48-hour observation and very slight erythema was noted at all treated skin sites at the 72-hour and 7-day observation. Very slight oedema was noted at all heated skin sites one hour after patch removal and persisted at the 24, 48 and 72-hour observations and at one treated skin site at the 7-day observation. Loss of skin elasticity was noted at all treated skin sites at the 48, 72-hour and 7-day observations. All treated skin sites appeared normal at the 14-day observation. Yellow/orange staining of the treatment site was noted during the study.

EYE IRRITATION

An in vivo study was performed to assess the irritation of the test material to the eye of rabbits. The method followed the OECD Guidelines 405. The test material produced scattered or diffuse to opalescent corneal opacity, iridial inflammation and severe conjunctival irritation. Vascularisation, with a localised ingrowth of vessels for 2 mm to 3 mm in length, was noted in one treated eye at the 7, 14 and 21 day observations. Orange coloured staining of the cornea and conjunctivae was noted in all treated eyes. The staining did not affect evaluation of ocular effects. Orange coloured staining of the fur around all treated eyes was noted throughout the study. The vascularisation noted in one treated eye at the 21-day observation was considered to be indicative of irreversible ocular damage. Therefore, test material was judged as to be capable to cause serious eye damage.

It should be considered that the lot tested in the experiment above described contains sodium hydroxide as impurity, which is a known corrosive agent (listed in annex VI of CLP Regulation (EC) No 1272/2008 and classified as Skin Corr 1A, H314). the content of sodium hydroxide was in the range of 0.5 - 2 %, therefore in a range sufficient to contribute to the effects recorded.

In order to clarify the eye irritation potential of the substance, an in vitro test was run, using the Isolated Chicken Eye Test (ICET) system. Under test conditions, the substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 1xII, 1xIV. Positive and negative controls showed the expected results, therefore, the experiment was considered to be valid. According to the guideline OECD 438, the substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category.

The results fell in the "grey area"; however, on the basis of which a prediction cannot be made, the severity of the effects recorded suggests that the substance is improbably able to cause eye damage.

DISCUSSION AND CONCLUSION

[details about the irritating potential conclusion are given in the document attached]

The substance was tested for skin and eye irritation potential, using for in vivo and in vitro tests two batches characterized by a different analytical profile: in the case of batch used for both the skin/eye irritation in vivo tests, the composition include an impurity able to impact the test results.

In order to evaluate the actual irritating potential of the substance, in particular in the case of eyes, a review of all the available information has been performed.

Although the Acid Yellow 236 content of batch used in in vivo tests is lower than the content of batch used in the in vitro ones, the effects recorded in the studies run in vivo are more severe than those reported in the tests run in vitro, both in the cases of skin and eye irritation; thus, it can be concluded that the impurity contributed to the effects recorded.

In conclusion, Acid Yellow 236 is not skin irritating; however, it can be classified as Eye Irrit. 2 (H319), according to the CLP Regulation (EC) 1272/2008.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.2 Skin corrosion/irritation section, skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

Based on the available information, the sustance is not skin irritating.

According to the CLP Regulation (EC) No 1272/2008, serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application.

Based on the available information, the sustance can be considered as able to cause serious eye irritation.

 

In conclusion, Acid Yellow 236 is not skin irritating; however, it can be classified as Eye Irrit. 2 (H319), according to the CLP Regulation (EC) 1272/2008.