Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Endpoint summary

Description of key information

Skin

In an in vitro skin irritation assay (reconstructed human epidermis) according to OECD 439, a mean cell viability of 104 % was determined. The test item is considered to be non-irritant to skin.

Eye

In an ex vivo assay in isolated chicken eyes (ICE) according to OECD guideline 438, the overall ICE class was 1xI, 1xII, 1xIII. The overall in vitro classification is neither UN GHS Classification Category I nor No Category. Thus, the test item has been categorized as “No prediction can be made”.

In a follow-up in vitro eye irritation test (RhCE) according to OECD guideline 492, a relative cell viability of 99% was determined, indicating no eye irritation potential under the applied testing conditions. Thus, the test item is considered as non-irritant to eye (UN GHS No Category).

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:

31 August - 10 October, 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:

28 July, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), 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:

2009 February

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiration date: 29 April 2020
Storage: Room temperature (15-25°C)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

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).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model.
- Tissue batch number: 16-EKIN-037
- Date of initiation of testing: 2016-09-14

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the EpiSkinTMSM units were rinsed thoroughly with approximately 25 mL 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
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: incubation in distilled water followed by freezing.
- N. of replicates: 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating/corrosive 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 substance is considered to be non-irritant to skin if 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

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 %

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours ± 1 hour

Number of replicates:

3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The non-specific MTT reduction (NSMTT) was determined to be 0.017 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: As the test item has an intrinsic colour (dark black), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.013. The Non Specific Colour % (NSC %) was calculated as 1.5 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Summary of neg. and pos. control results

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	1.018	117
	2	0.823	95
	3	0.765	88
	mean	0.869	100
	standard deviation (SD)		15.22
Positive Control: SDS (5 % aq.)	1	0.167	19
	2	0.168	19
	3	0.072	8
	mean	0.136	16
	standard deviation (SD)		6.34

Table 2: OD values and viability percentages of the test item (including corrected values)

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Leuco Sulfur Blue 11	1	1.059	1.059	122	122
	2	0.811	0.811	93	93
	3	0.845	0.844	97	97
	mean	0.905	0.905	104	104
	standard deviation (SD)			15.49	15.49

TOD_TT :true MTT metabolic conversion

Table 3: OD values of additional controls for MTT-interacting test item

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.065
	2	0.078
	3	0.079
	mean	0.074
Test item treated killed tissues: Leuco Sulfur Blue 11	1	0.066
	2	0.059
	3	0.099
	mean	0.075

 

 

Table 4: OD values and NSC % of additional control

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
Leuco Sulfur Blue 11 (test item treated tissueswithout MTT incubation)	1	0.008	1.5
	2	0.019
	mean	0.013

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo skin irritation assay (recontructed human epidermis) according to OECD 439, a mean cell viability of 104 % was determined. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

An in vitro skin irritation assay (recontructed human epidermis, RhE) has been performed according to the OECD Test Guideline No. 439, to determine the skin irritation potential by measurement of the cytotoxic effect of the test item, as reflected in the MTT assay. Disks of EPISKIN (three units) were treated with 10 mg of the test item and incubated for 15 minutes at room temperature. Exposure to the test item was terminated by rinsing with PBS 1x solution. The tissues were then incubated at 37 °C for 42 hours at 37 °C. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. 10 µL SDS (5 % aq.) or 10 µL 1x PBS treated tissues (three replicatrees each) 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 (dark black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference. Two killed treated tissues were used to avoid a possible double correction for colour interference. Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid

In this in vitro skin irritation test using the EPISKIN model, cell viability was not significantly reduced (corrected value (mean relative viability: 104 %) after test item treatment compared to the negative control. Therefore the test item was considered to be not irritanting to the skin.

.

 

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:

23 August - 22 September, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July, 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:

Expiration date: 29 April 2020
Storage: Room temperature (15-25°C)

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: All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Temperature and transport conditions of ocular tissue: 2 h at ambient temperature (19.4 ºC to 20.3 ºC)
- indication of any existing defects or lesions in ocular tissue samples: 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.
- Indication of any antibiotics used: not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg/eye

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 minutes

Number of animals or in vitro replicates:

3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.

Details on study design:

isolated chicken eye test (ICET)
Removal of test item: 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 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.
The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 minutes of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.
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 .

Tool used to assess score: hand-slit lamp/biomicroscope/fluorescein
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.
Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current).

After collection, 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).
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. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
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. 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.


SELECTION AND PREPARATION OF ISOLATED EYES
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. 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. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was 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 5 drops/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, 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. If the selected eyes were appropriate for the test, 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. 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 2%) were observed in the eyes, 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.

NUMBER OF REPLICATES
3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.

NEGATIVE CONTROL USED
30 µL NaCl (9 g/L saline)

POSITIVE CONTROL USED
30 mg Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 mg test item, 10 s

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes
- Damage to epithelium based on fluorescein retention: Yes
- Swelling: measured with optical pachymeter on a slit-lamp microscope, yes

SCORING SYSTEM:
- Mean corneal swelling (%) : Yes
- Mean maximum opacity score : Yes
- Mean fluorescein retention score at 30 minutes post-treatment : Yes

DECISION CRITERIA: Tthe decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: over all ICE score: 1xI; 1xII; 1xIII

Remarks:

Overall in vitro classification is neither UN GHS Classification Category I nor No Category; test item has been categorized as “No prediction can be made”.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Table 2: Summary of the results test item)

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	3%	I
Mean maximum corneal swelling at up to 240 min	4%	I
Mean maximum corneal opacity	1.7	III
Mean fluorescein retention	1.3	II
Other Observations	None
Overall ICE Class^1	1xI, 1xII, 1xIII

Table 3: Positive Control (Imidazole)

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	25%	III
Mean maximum corneal swelling at up to 240 min	33%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in two eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	3xIV

The positive control Imidazole was classed as corrosive/severely irritating, UNGHS Classification: Category 1.

 

Table 4: Negative Control: (NaCl, 9 g/L saline))

Observation	Value	ICE Class^1
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	0.5	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class^1	3xI

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study. Positive and negative control values were within the corresponding historical control data range.

Interpretation of results:

study cannot be used for classification

Conclusions:

In an ex vivo assay in isolated chicken eyes (ICE) according to OECD guideline 438, the overall ICE class was 1xI, 1xII, 1xIII. The overall in vitro classification is neither UN GHS Classification Category I nor No Category. Thus, the test item has been categorized as “No prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICE Test) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test was performed according to OECD guideline 438. 30 mg/ of the test item or positive control or 30 µL of the negative control (NaCl, 9 mg/L saline), repectively, were applied to the cornea. Three test item treated eyes, three positive eyes and one negative control eye were used in this study.

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. Tested corneas were evaluated pre-treatment and at 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 time points above. The overall ICE class for the test item was 1xI, 1xII, 1xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

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

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 February - 02 June, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiration date: 29 April 2020
Storage: Room temperature (15-25°C)

Species:

human

Strain:

other: EpiOcular human cell construct

Remarks:

keratinocytes

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability :
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that also water insoluble materials may be tested. Prior to use, each plate (6, 12, and 24-well) and its cover will be uniquely identified with a permanent marker by a plate number and/or test item number. The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test item treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).

Quality Control:
The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue 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 Triton X-100 (100 μL of 0.3% (v/v) Triton X-100). A certificate of quality as provided by the supplier is annexed to this report.
Number of Replicate Wells.
The condition of the cooler boxes was checked, because the frozen cooler box can guarantee that the temperature of kit was between 2-8°C during the transport.
The cooler boxes were frozen and they were not in direct contact with any of the 24 well plates containing the EpiOcular™ tissues (OCL-200).
The kit was found to be in good order at reception.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes, at standard culture conditions

Number of animals or in vitro replicates:

2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.

Details on study design:

- Details of the test procedure used :
The tissues were equilibrated to room temperature for about 15 minutes, while the Assay Medium was pre-warmed to 37±1 °C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37 °C in an incubator with 5±1% CO2, 90±10% humidified atmosphere for one hour in the Assay Medium, than the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours). During the pre-treatement the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.

- RhCE tissue construct used, including lot number
EpiOcular™ (OCL-200-EIT), MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, 821 05 Bratislava, Slovakia; Lot No. 23779

- Doses of test chemical and control substances used
50 mg of the test item or a volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions
After rinsing, the tissues were transferred to and immersed in 5 mL RT Assay Medium in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at RT.
The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
As the test item has an intrinsic colour (dark black), further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues. In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer and has an intrinsic colour (dark black). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed

- Number of tissue replicates used per test chemical and controls
2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.

- Wavelength used for quantifying MTT formazan
570 nm. 96-well plate spectrophotometer

- Description of the method used to quantify MTT formazan
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, 90±10% humidified atmosphere. Inserts were removed from the 24-well plate after 3 hours ± 10 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm. To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically. Following the formazan extraction, 200 µL sample(s) from each tube (preferably 2×200 µL if possible) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 µL).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2, further testing with other test methods will be required to decide on its final classification. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

- Complete supporting information for the specific RhCE tissue construct used
see "Any other information on materials and methods"

- Acceptable variability between tissue replicates for positive and negative controls and acceptable variability between tissue replicates for the test chemical
The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is:
- 30 minute exposure: below 50% of control viability (liquids)
- 6 hours exposure: below 50% of control viability (solids)
The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: relative viability %

Run / experiment:

mean

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: optical density

Run / experiment:

mean

Value:

1.716

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Acceptance criteria:

The mean OD value of the two negative control tissues was: 1.677

The positive control result showed 26 % viability at 6 hours exposure.

The difference of viability between the two tissue replicates 0.7% to 8.6%

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Possible direct MTT reduction with test item:

As the test item has an intrinsic colour (dark black), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.

The non-specific MTT reduction (NSMTT) was determined to be 3.110 %. As the NSMTT were below 60 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

Colouring potential of test item:

As the test item has an intrinsic colour (dark black), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.014. The Non Specific Colour % (NSC %) was calculated as 0.84 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Cell viability

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

 

Table 3: OD values and viability percentages of the controls:

Controls	Optical Density (OD)		Viability (%)	Δ%
Negative Control: Sterile deionized water	1	1.740	104	7.5
	2	1.614	96
	mean	1.677	100
Positive Control: Methyl acetate	1	0.509	30	8.6
	2	0.364	22
	mean	0.437	26

 

Table 4: OD values and viability percentages of the test item (including corrected values):

Test Item	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)	Δ%
Leuco Sulfur Blue 11	1	1.645	1.593	98	95	8.4
	2	1.787	1.735	107	103
	mean	1.716	1.664	102	99
	standard deviation (SD)			5.96	5.96

 

Table 5: OD values of additional controls for MTT-interacting test item:

Additional controls	Optical Density (OD)
Negative control killed tissues: Sterile deionized water	1	0.042
	2	0.193
	mean	0.118
Test item treated killed tissues: Leuco Sulfur Blue 11	1	0.136
	2	0.204
	mean	0.170

 

 

Table 6: OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSCliving %)	Δ%
Leuco Sulfur Blue 11	1	0.008	0.84	0.7
	2	0.020
	mean	0.014

Remark:Δ%: The difference of viability between the two relating tissues

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro eye irritation test (RhCE) according to OECD guideline 492, a relative cell viability of 99% was determined, indicating no eye irritation potential under the applied testing conditions.

Executive summary:

The eye irritating potential f the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. Two replicates of EpiOcular™ tissues were treated with test item (50 mg) , positive control (methyl acetate, 50 µL) or negative control (sterile deionized water, 50 µL), respectively and incubated for 6 hours (± 15 min) at standard culture conditions (37°C in an incubator with 5 % CO2, 90±10% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution at 37°C. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (dark black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 99 %). Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from thisin vitroeye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is considered as non-irritant to eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin

An in vitro skin irritation assay (recontructed human epidermis, RhE)has been performedaccording to the OECD Test Guideline No. 439,to determine the skin irritation potential by measurement of the cytotoxic effect of the test item, as reflected in the MTT assay.Disks of EPISKIN (three units) were treated with 10 mg of the test item and incubated for 15 minutes at room temperature. Exposure to the test item was terminated by rinsing with PBS 1x solution. The tissues were then incubated at 37 °C for 42 hours at 37 °C. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. 10 µLSDS (5 % aq.) or 10 µL 1x PBS treated tissues (three replicatrees each) 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 (dark black), therefore two additional test item treated tissues were used for the non-specific OD evaluation.The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference. Two killed treated tissues were used to avoid a possible double correction for colour interference.Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid

In this in vitro skin irritation test using the EPISKIN model, cell viability was not significantly reduced (corrected value (mean relative viability: 104 %) after test item treatment compared to the negative control. Therefore the test item was considered to be not irritanting to the skin.

Eye

The objective was to determine a possible eye irritating potential of the test substance by in vitro studies. A single in vitro assay may not always be sufficient, using the currently available methods, to cover the full range of eye irritating potential. Consequently, two in vitro assays were performed in a weight of evidence approach: The EpiOcular Eye Irritation (RhCE) Test according to OECD guideline 492 and the Isolated Chicken Eye Test (ICE Test) according to OECD guideline 438.

ICE:

The purpose of this Isolated Chicken Eye Test (ICE Test) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test was performed according to OECD guideline 438. 30 mg/ of the test item or positive control or 30 µL of the negative control (NaCl, 9 mg/L saline), repectively, were applied to the cornea. Three test item treated eyes, three positive eyes and one negative control eye were used in this study.

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.Tested corneas were evaluated pre-treatment and at 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 time points above. The overall ICE class for the test item was 1xI, 1xII, 1xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

According to the guideline OECD 438, the overall in vitro classification of the test item is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the OECDguideline438, the test item has been categorized as “No prediction can be made”. Consequently, a furhter in vitro was initiated to allow a final conclusion on the eye irritiation potential of the test item (see below).

RhCE:

The eye irritating potential f the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. Two replicates of EpiOcular™ tissues were treated with test item (50 mg) , positive control (methyl acetate, 50 µL) or negative control (sterile deionized water, 50 µL), respectively and incubated for 6 hours (± 15 min) at standard culture conditions (37°C in an incubator with 5 % CO2, 90±10% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution at 37°C. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (dark black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 99 %). Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from thisin vitroeye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is considered as non-irritant to eye (UN GHS No Category).

In conclusion, based on the results of the performed assays, the test item is considered as non-irritant to eye.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the test item is not considered to be classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/52.