[No public or meaningful name is available]

Endpoint summary

Description of key information

Not skin irritating

Not eye irritating

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:

February from the 12th to the 14th, 2014

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:

26 July 2013.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

Version 1.8 (February 2009)

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

Source species:

human

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 no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France.
- Tissue batch number: 14-EKIN-004
- Cell source: adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994).
- Manufacture: EpiSkinTMSM kits are manufactured according to defined quality assurance procedures (certified ISO 9001).
- Safety: all biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
- Storage: the EpiSkinTMSM units were kept in their packaging at room temperature until the preincubation was started. The maintenance and assay medium were stored at 2-8 °C.

REMOVAL OF TEST MATERIAL: after the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with PBS 1x solution to remove all of the test material from the epidermal surface. 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).

CONTROLS
- Replicates: three replicates were used.
- Application: 10 μl positive control or negative control (1x PBS) was 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT stock solution: MTT was dissolved to a final concentration of 3 mg/ml in saline buffer (PBS).
- Storage of stock solution: the obtained stock solution can be stored in refrigerator (2-8 °C), protected from light up to 15 days.
- MTT concentration: MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/ml. The obtained solution was used within one hour.
- Incubation time: an amount of 10 mg test item was added to 2 ml MTT 0.3 mg/ml solution and mixed. The mixture was incubated for three hours at 37 °C protected from light and then any colour change observed.
- Interation evaluation: if the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).
- MTT test after 42 hours incubation: after the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells and then incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO2 protected from light.

ADDITIONAL CONTROL
- Non specific OD evaluation: one additional chemical-treated tissue was used for the non-specific OD evaluation.
- Ttreatment: the tissue followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium.
- Reading: OD reading was made following the same conditions as for the other tissues.

FORMAZAN EXTRACTION
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from the unit using a biopsy punch. 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 then incubated for about two hours at 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 MEASUREMENTS
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 using acidified isopropanol solution as the blank (6×200 μl).

ASSAY 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 should be ≤ 18.

INTERPRETATION OF RESULTS
According to EU classification, the irritancy potential of test items is predicted for distinguishing between R38 skin irritating and no-label (non-skin irritating) test substances OECD TG 404 & Method B.4 of Annex V to Directive 67/548/EEC. In the present study, the irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test substance is considered to be irritant to skin (R38), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied in its original form, no formulation was required.
The epidermal surface was first moistened with 10 μl deionised water and then 10 mg of the test item was applied evenly onto the skin. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.

Duration of treatment / exposure:

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

Duration of post-treatment incubation (if applicable):

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.

Number of replicates:

Three replicates.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

> 50

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item showed reduced cell viability in comparison to the negative control (mean value: 67 %). However 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.

MTT REDUCTION
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.

OD EVALUATION
As the test item has an intrinsic colour (brown), one additional chemical-treated tissue was used for the non-specific OD evaluation. The OD (measured at 570 nm) of this tissue was determined as 0.024. The Non Specific Colour % (NSC %) was calculated as 3.0 %.
Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.819, the positive control result showed 6 % viability, each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

OD values and viability percentages of the test item

	Optical Density (OD)		Viability (%)
Test item	1	0.593	72
	2	0.629	77
	3	0.428	52
	Mean	0.550	67
	standard deviation (SD)		13.07

OD values and NSC % of additional control

	Optical Density (OD)		Viability (%)
test item treated tissues without MTT incubation	1	0.024	3.0

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells.

Controls	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.764	93
	2	0.860	105
	3	0.833	102
	Mean	0.819	100
	standard deviation (SD)		6.02
Positive Control: SDS (5 % aq.)	1	0.067	8
	2	0.045	6
	3	0.037	5
	Mean	0.050	6
	standard deviation (SD)		1.91

Interpretation of results:

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

Conclusions:

Not irritating.

Executive summary:

The irritation potential of test item has been assayed by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. The experiment was conducted following the procedures outlined into the OECD guideline 439. 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. 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. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. The test item has an intrinsic colour (orange), one additional chemical-treated tissue was used for the non-specific OD evaluation. SDS (5 % aq.) and PBS treated epidermis were used as positive and negative controls respectively (three units / control). For each treated tissue viability was expressed as a percentage relative to negative control. The test item is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control. The test item showed reduced cell viability in comparison to the negative control (mean value: 67 %). However 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

In the EPISKIN model, test item resulted to be non Irritant (NI) [UN GHS: No Category].

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:

key study

Study period:

February the 17th. 2014

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

Qualifier:

according to guideline

Guideline:

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

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, but it was ground finely in a mortar and pestle before use in the study.

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Species of chicken: COBB 500
- Source: TARAVIS KFT. 9600 Sárvár. Hungary
- Head collection: collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current).
- Transport to laboratory: the heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.9 ºC to 20.5 ºC) during the transport.
- Source of eye: all eyes used in the assay were from the same groups of eyes collected on one specific day.
- Health check: the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item was applied in a volume of 0.03 g by powdering the entire surface of the cornea of isolated chicken eyes

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control eyes and one negative control eye were used in this study

Details on study design:

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.

EYE 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
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. Slight changes in thickness (0 % to 3 %) were observed in the eyes, a finding considered as normal when maintaining enucleated 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, taking care not to damage or touch the cornea with the application equipment.

POSITIVE CONTROL USED
The three positive control eyes were treated in as test item group with 0.03 g 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. The substance was applied in a volume of 0.03 g by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eyes.

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.
Test item was stuck on the corneas surface at 30 minutes after the post-treatment rinse. The gentle rinsing with additional 20 ml saline was performed after 30 minutes of observation. The cornea surfaces were totally cleared at 75 min after the post-treatment rinse.

OBSERVATION PERIOD
The control 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 ±5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

SCORING SYSTEM
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

The effects were divided into four categories for each endpoint:
I = none
II = slight
III = moderate
IV = severe

Cornea 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

Fluorescein 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

Irritation parameter:

in vitro irritation score

Remarks:

ICE Class

Run / experiment:

mean

Value:

1 - 2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In in vitro eye irritation study using the isolated chicken eyes, no ocular corrosion or severe irritation potential was observed. The test item was not a severe irritant.

Positive and negative control values were within the corresponding historical control data ranges.

Test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2 %	I
Mean maximum corneal swelling at up to 240 min	3 %	I
Mean maximum corneal opacity	0.7	II
Mean fluorescein retention	1.2	II
Overall ICE Clas		1xI, 2xII

Other Observations: the test item was stuck on the cornea surfaces at 30 minutes after the post-treatment rinse. The cornea surfaces were totally cleared at 75 min after the post-treatment rinse.

Negative control

bservation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Overall ICE Clas		3xI

Other Observations: none.

Positive Control: Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	17 %	III
Mean maximum corneal swelling at up to 240 min	28 %	III
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	2.8	IV
Overall ICE Clas		1xIII, 2xIV

Other Observations: cornea opacity score 4 was observed in three eyes at 30 minutes after the posttreatment rinse.

Interpretation of results:

other: substance does not cause ocular corrosion or severe irritation

Conclusions:

Test item is not classified as an ocular corrosive or severe eye irritant.

Executive summary:

The purpose of the Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes.The experiment was conducted following the procedures outlined into the OECD guideline 438. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. The damage by the test substance was assessed by determination of corneal swelling, opacity, fluorescein retention and morphological effects. These parameters were evaluated pre-treatment and starting 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. The test item and Imidazole (positive control) were applied in a volume 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.

The substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Conclusion

Test item is not classified as an ocular corrosive or severe eye irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March from the 11th to the 19th, 2014

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 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2nd October 2012

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S & K-LAP Kft.
- Age at study initiation: adult rabbits, 11 weeks old.
- Weight at study initiation: 2952 - 2992 g.
- Housing: 8 (E building). Animals were housed individually in metal cage.
- Diet: the animals received CRLT/ny rabbit diet produced by Szindbád Kft., ad libitum.
- Water: animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed.
- Acclimation period: 5 days in first animal and 6 days in second and third animal.
- Animal health: only animals in acceptable health condition were used for the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C.
- Humidity: 30-70 %.
- Air changes: 10-15 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g of the test item was used for the study in pure state, in a single dose.

Duration of treatment / exposure:

The eyes of the test animals were not washed out 24 hour after test item application, because the test item had been removed from the eye of test animal by physiological mechanisms.

Observation period (in vivo):

1 week

Number of animals or in vitro replicates:

3 males

Details on study design:

TEST SYSTEM
In the first step an initial test was performed using one animal. The test item was placed into the conjunctival sac of the left eye. The eyelids were held closed gently for about one second to prevent any loss of the test item. In the initial test no irritant effect was observed 24 hours after the treatment. The confirmatory test was conducted using a second and third animal.

TOPICAL ANESTHETICS
Approximately sixty minutes prior to test item application systemic analgesics (dose: 0.01 mg/kg) was administered subcutaneously.
Five minutes prior to test item application topical ocular anesthetic (dose: one drop) was applied to each eye. The eye of each animal that was not be treated with a test item, but which was treated with topical anesthetics, served as a control.

SCORING SYSTEM:
The eyes were examined at 1, 24, 48 and 72 hours, then 1 week after the application. The duration of the observation period was sufficient for the statement of reversibility or irreversibility of changes.
Any clinical signs of toxicity or signs of pain and/or distress were recorded at least twice daily, with a minimum of 6 hours between observations.

The eye irritation scores were evaluated according to the scoring system by Draize (1979) and OECD 405 (02 October 2012).

Irritation parameter:

cornea opacity score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 1

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: 3/3

Time point:

24/48/72 h

Score:

< 2

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

One hour after treatment some hyperemic blood vessels (score 1) were observed in animal No.: 00455. The amount of discharge was different from normal (score 1) in two animals (No.: 00455, 00454). However, other sign as yellow discoloured cornea and eye periphery was detected in all animals.
24 hours after treatment other sign as yellow discoloured cornea and eye periphery was detected in all animals. There was no any eye irritation effects were observed.
48 hours after treatment the state of animals did not change.
72 hours after treatment other sign as yellow discoloured cornea and eye periphery was recorded in animal No.: 00455.
1 week after treatment all animals were free of symptoms, so the study was finished.

During the study the control eyes of animals were symptom-free.

No systemic toxicity was observed on the day of the treatment and during the 1-week observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in two animals (No.: 00455, 00454). This symptom was detected on the treatment day 1 hour after the treatment.

Individual scores for ocular irritation

After the treatment	Animal N.	Score irritation
		R	CH	D	OD	OE	R	Control eye	Other signs
1 h	00455	1	0	1	0	0	0	0	S
	00452	0	0	0	0	0	0	0	S
	00454	0	0	1	0	0	0	0	S
24 hrs	00455	0	0	0	0	0	0	0	S
	00452	0	0	0	0	0	0	0	S
	00454	0	0	0	0	0	0	0	S
48 hrs	00455	0	0	0	0	0	0	0	S
	00452	0	0	0	0	0	0	0	S
	00454	0	0	0	0	0	0	0	S
72 hrs	00455	0	0	0	0	0	0	0	S
	00452	0	0	0	0	0	0	0	-
	00454	0	0	0	0	0	0	0	-
1 wk	00455	0	0	0	0	0	0	0	-
	00452	0	0	0	0	0	0	0	-
	00454	0	0	0	0	0	0	0	-

R = Redness; OD = Opacity degree of density; CH = Chemosis; OE = Extension of opaque area; D = Discharge; S = Yellow discoloured cornea and eye periphery.

Body weight data

Animal N.	Beginning of study (g)	At the end of study (g)	Body weight gain (g)
00455	2992	3190	198
00452	2952	3110	158
00454	2952	3166	214

Interpretation of results:

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

Conclusions:

Not irritating.

Executive summary:

The acute eye irritation study with test item was performed in New Zealand White rabbits, according to the OECD guideline 405. The irritation effect of the test item was scoring according to the Draize method (Draize, 1959; OECD Guideline 405, 2012). The test item was placed into the conjunctival sac of left eye of each animal. The untreated right eye served as control. 0.1 g of the test item was used for the study in pure state, as a single dose. The eyes of the test animals were not washed out.

Treated and control eyes were examined at 1, 24, 48 and 72 hours, then 1 week after the application.

1 hour after the single application of test item into the eye of the rabbits, slight redness and discharge occurred. However, the test item dyed the cornea and eye periphery yellow. 24 hours after treatment, other sign as yellow discoloured cornea and eye periphery was observed. 1 week after treatment all animals were free of symptoms, so the study was finished.

During the study the control eyes of animals were symptom-free.

No systemic toxicity was observed on the day of the treatment and during the 1-week observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in two animals. This symptom was detected on the treatment day 1 hour after the treatment.

Conclusion

Not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

The irritation potential of test item has been assayed by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN reconstituted human epidermis. The experiment was conducted following the procedures outlined into the OECD guideline 439. The test item showed reduced cell viability in comparison to the negative control (mean value: 67 %). However 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 and the is considered to be non irritant.

EYE IRRITATION

The test item has been firstly assayed in an in-vitro Isolated Chicken Eye Test; the purpose of the experiment was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Because the determined physical-chemical properties and the chemical reactivity of test item did not reveal potential corrosion effects to the eye and because the in-vitro chicken eye corrosion/irritation study with the test compound did not indicate corrosion/severe irritation potential, a further in-vivo testing for eye irritation/corrosion was carried out.

The acute eye irritation study with test item was performed in New Zealand White rabbits, according to the OECD guideline 405. 1 hour after the single application of test item into the eye of the rabbits, slight redness and discharge occurred. However, the test item dyed the cornea and eye periphery yellow. 24 hours after treatment, other sign as yellow discoloured cornea and eye periphery was observed. 1 week after treatment all animals were free of symptoms, so the study was finished. No systemic toxicity was observed on the day of the treatment and during the 1-week observation period. The body weight of animals corresponded to their species and age.

It has been concluded that the substance can be considered as non irritant.

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. However, in vitro alternatives that have been validated and accepted may also be used to help make classification decisions.

Yellow LF 6881 has been assayed by validated and accepted in vitro EpiSkin reconstituted human epidermis model. The average viability of treated tissues was 67 %, i.e. viability was higher than 50 %, thus the effect of the test substance was judged as negative.

 

According to the CLP Regulation (EC) No 1272/2008, substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) if, when applied to the eye of an animal, a substance produces at least in 2 of 3 tested animals, a positive response of corneal opacity ≥ 1 and/or iritis ≥ 1, and/or conjunctival redness ≥ 2 and/or conjunctival oedema (chemosis) ≥ 2, calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

The mean values from gradings at 24, 48 and 72 hours recorded in the key study were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all animals in the key study.

 

In conclusion, the substance does not meet the criteria to be classified for skin/eye irritation, according to the CLP Regulation (EC) No 1272/2008.