1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Endpoint summary

Description of key information

Skin irritation / corrosion (in vitro) (Buda 2021)

Under the conditions of this study the test material was not irritating.

 

Eye irritation (ex vivo) (Buda 2021)

Under the conditions of this study the test material was found to be non-irritating to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2021 to 05 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 131 (09 June 2012): EpiSkinTM Skin Irritation Test 15 min – 42 hours

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiSkin™ Small Model (EpiSkin™SM)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: See below

Source strain:

other: Adult human derived

Details on animal used as source of test system:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN Laboratories Lyon, France, is a three-dimensional human epidermis model. 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). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Supplier: EPISKIN Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 21-EKIN-022
Expiry date: 07 June 2021

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 ON THE 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
- Tissue batch number(s): 21-EKIN-022
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 03 June 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.9-23.7 °C)
- Temperature of post-treatment incubation (if applicable): 37±1 °C

NUMBER OF REPLICATE TISSUES: Three replicates were used for the test item and positive and negative controls.
Additionally, 2 replicates of colour controls (NSCliving), 2 replicates of non-specific colour control (NSCkilled), 3 killed test item treated tissues and 3 killed negative control treated tissues were used for the MTT evaluation.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL 1x PBS (phosphate buffered saline) solution

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): Place the living epidermis in a 12 well plate with 2 mL of distilled water (replacing the culture medium). Incubate at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere for 48 h +/- 1 hours. At the end of the incubation, discard the water.
- N. of replicates : 3 killed test item treated tissues and 3 killed negative control treated tissues
- Method of calculation used:
Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 5 (Test Item, Positive and negative control, Additional controls for MTT direct interacting chemicals, Additional controls for dyes and chemicals able to colour the tissue and Additional controls for non-specific colour in killed tissues)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritating to skin if the viability after 15 minutes exposure is less than 50% of the negative control

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: yes, for the non-specific OD evaluation (NSCliving), and also to detect and correct for test item interference with the viability measurement

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 10 μL (1x PBS)

POSITIVE CONTROL
- Amount(s) applied: 10 μL (SDS 5%)

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 h)

Number of replicates:

In this assay 3 replicates of test item, 3 replicates of negative control, 3 replicates of positive control, 2 replicates of colour controls (NSCliving), 2 replicates of non-specific colour control (NSCkilled), 3 killed test item treated tissues and 3 killed negative control treated tissues were used for the MTT evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 runs

Value:

75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

corrected relative viability

Run / experiment:

Mean of 3 rubs

Value:

71

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: Not significant
- Colour interference with MTT: Not significant

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was 1.310 and the % tissue viability was 100%.
- Acceptance criteria met for positive control: Yes. The mean OD value obtained for the positive control was 0.059 and this result corresponds to 4 % viability.
- Acceptance criteria met for variability between replicate measurements: Yes. 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.

OD values and viability percentages of the positive and negative controls and test material

Substance	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Negative control: 1x PBS	1	1.408	-	108	-
	2	1.311	-	100	-
	3	1.210	-	92	-
	mean	1.310	-	100	-
	standard deviation (SD)			7.57	-
Positive Control: SDS (5 % aq.)	1	0.049	-	4	-
	2	0.048	-	4	-
	3	0.079	-	6	-
	mean	0.059	-	4	-
	standard deviation (SD)			1.32	-
Test Material	1	0.973	0.918	74	70
	2	1.158	1.102	88	84
	3	0.826	0.771	63	59
	mean	0.986	0.930	75	71
	standard deviation (SD)			12.69	12.69

 

INDICATOR FOR POTENTIAL FALSE VIABILITY

Possible direct MTT reduction with test material - As the test material has an intrinsic colour (red), the check-method for possible direct MTT reduction with test material was not completed for the following reason: if the test material has dark colour (red, green, blue ect.), the additional controls were used automatically. This step prevents the false viability which is the possible interaction with MTT, and it occurs if the test material cannot be removed totally from the surface of the tissues. In this situation the direct interaction with MTT is defined based on the OD results of additional controls. The non-specific MTT reduction (NSMTT) was determined to be 4 %. As the NSMTT was below 50 %, the true MTT metabolic conversion and the correction of viability percentages were undertaken.

Colouring potential of test material - The test material has an intrinsic colour (red). If the test material is not white, off white, almost white and/or colourless the additional controls are automatically used and based on the OD results of additional controls the Non Specific Colour % (NSCliving %) is determined. This step prevents the false viability, which can possibly be caused by the stained surface of the tissues by the test material. Two additional test material-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.046. The Non Specific Colour % (NSCliving %) was calculated as 3 % (below 5 %). Therefore, additional data calculation was not necessary. A false estimation of viability can be precluded.

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

In this in vitro skin irritation test using the EPISKIN model, the test material did not show significantly reduced cell viability in comparison to the negative control (mean corrected relative viability value: 71 %). All obtained test material viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore, the test material was considered to be non-irritant to skin under the conditions of this study.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B. 46 in accordance with GLP using the EPISKIN Model.

Using this method, a small amount of the test material was applied to the surface of a three-dimensional human epidermis model (EpiSkin) and the optical density (OD) measured after an incubation period, from which the relative cell viability could be calculated. Additionally, positive and negative controls were performed, as well as controls for possible MTT-interacting items and chemicals able to colour the tissue.

Since the % relative tissue viability was calculated to be >50% for the test item, under the conditions of this study the test material was not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2021 to 19 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test material was not ground, because the contact between the test item and the surface of the cornea was considered suitable without the grinding process.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

COLLECTED EYES
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue: The heads were transported to the test facility at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 - 20.1 °C) in first experiment and the additional experiment during the transport.
- Time interval prior to initiating testing: None
- Indication of any existing defects or lesions in ocular tissue samples: Only healthy eyes were used
- Indication of any antibiotics used: Not specified
- Selection and preparation of corneas:
Eyes selection - After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. Cornea integrity was checked by applying one small drop of fluorescein 2% (w/v) solution 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 a 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. 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.
- Quality check of the isolated corneas: 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. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate numbers of eyes were 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, equaling 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg per eye

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

The observation times were at 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

Number of animals or in vitro replicates:

3 eyes were exposed to the test material
3 eyes were exposed to the positive control
1 eye was exposed to the negative control

Details on study design:

EYE ACCLIMATISATION
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.

TEST PROCEDURE
The baseline assessments - 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%) was observed in the eyes during the first experiment. The changes in thickness was not observed in the eyes during the additional experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

Treatment - After the zero reference measurements in each experiment, one out of three test material 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 material was applied onto the center of the cornea. The test material was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test material, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test material treated eye. The three positive control eyes were treated in a similar way with 0.03 g of Imidazole. 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. Based on the results of the experiment the test material showed negative outcome (GHS NC), consequently additional experiment was necessary to confirm or discard the negative outcome. All the steps in the additional experiment were the same as in the first experiment was.

Test material removal - 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 material 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 test material was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and the additional experiments. Gentle rinsing with 20 mL saline was performed in all test material treated eyes after the 30, 75, 120 and 180 minutes of observation. The test material treated cornea surfaces were not totally clear at 240 minutes after the post-treatment rinse in the first and the additional experiments. The positive control item was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and the additional experiments. Gentle rinsing with 20 mL saline was performed in all positive control treated eyes after the 30, 75, 120 and 180 minutes of observation. The positive control treated cornea surfaces were not totally clear at 240 minutes after the post-treatment rinse in the first and the additional experiments.

Observation and assessment of corneal effects - 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.

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.

Histopathology - Histopathology of the corneas was not performed. Corneas are discarded 2 months after the final report.

Irritation parameter:

corneal swelling 

Remarks:

(mean maximum at up to 75 min) (in %)

Run / experiment:

First experiment

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

corneal swelling 

Remarks:

(mean maximum at up to 240 min) (in %)

Run / experiment:

First experiment

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

cornea opacity score

Remarks:

(mean maximum)

Run / experiment:

First experiment

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

fluorescein retention score

Remarks:

(mean)

Run / experiment:

First experiment

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

corneal swelling 

Remarks:

(mean maximum at up to 75 min) (in %)

Run / experiment:

Second experiment

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

corneal swelling 

Remarks:

(mean maximum at up to 240 min) (in %)

Run / experiment:

Second experiment

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

cornea opacity score

Remarks:

(mean maximum)

Run / experiment:

Second experiment

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

fluorescein retention score

Remarks:

(mean)

Run / experiment:

Second experiment

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

Test material: In this study, the test material did not cause ocular corrosion or severe irritation in the enucleated chicken eyes in either the first or second experiments. The overall ICE classes were thrice I (based on corneal swelling of 2 % within 240 minutes, corneal opacity score of 0.5 and fluorescein retention of 0.5) in the first experiment and the overall ICE classes were twice I (based on corneal swelling of 1 % within 240 minutes and fluorescein retention of 0.5) and once II (based on the corneal opacity score of 0.8) in the additional experiment.

Positive and negative controls: The overall ICE classes for the positive control were 3xIV in the first experiment and 3xIV in the additional experiment. Therefore, the positive control was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1. The overall ICE classes for the negative control were 3xI in the first experiment and 3xI in the additional experiment. Therefore, the negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this experiment. Furthermore, the three endpoints of the positive and the negative controls were in the historical control range. So, the positive and negative controls showed the expected results in the first and in the additional experiment. The experiments were considered to be valid.

Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change in the first experiment

	Test Material		Positive Control		Negative Control
Observation	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min (%)	2	I	33	IV	2	I
Mean maximum corneal swelling at up to 240 min (%)	2	I	34	IV	2	I
Mean maximum corneal opacity	0.5	I	4.0	IV	0.5	I
Mean fluorescein retention	0.5	I	3.0	IV	0.0	I
Other Observations	None		Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.		None
Overall ICE Class	3 x I		3 x IV		3 x I

 

 

Mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change in the additional experiment

	Test Material		Positive Control		Negative Control
Observation	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min (%)	1	I	35	IV	0	I
Mean maximum corneal swelling at up to 240 min (%)	1	I	39	IV	0	I
Mean maximum corneal opacity	0.8	II	4.0	IV	0.0	I
Mean fluorescein retention	0.5	I	3.0	IV	0.0	I
Other Observations	None		Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.		None
Overall ICE Class	2 x I, 1 x II		3 x IV		3 x I

 

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

Under the conditions of this study the test material was found to be non-irritating to the eye.

Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions.

The in vitro eye irritation study was performed in isolated chicken’s eyes. Three eyes were treated with 30 mg of the test material. The three positive control eyes were treated in a similar way with 30 mg of powdered imidazole. The negative control eye was treated with 30 μL of saline. After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

In the first experiment, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by an additional experiment. The second run confirmed the negative results.

The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Under the conditions of this study the test material was found to be non irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion (in vitro) (Buda 2021)

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B. 46 in accordance with GLP using the EPISKIN Model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Using this method, a small amount of the test material was applied to the surface of a three-dimensional human epidermis model (EpiSkin) and the optical density (OD) measured after an incubation period, from which the relative cell viability could be calculated. Additionally, positive and negative controls were performed, as well as controls for possible MTT-interacting items and chemicals able to colour the tissue.

Since the % relative tissue viability was calculated to be >50% for the test material, under the conditions of this study the test material was not irritating.

 

Eye irritation (ex vivo) (Buda 2021)

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The in vitro eye irritation study was performed in isolated chicken’s eyes. Three eyes were treated with 30 mg of the test material. The three positive control eyes were treated in a similar way with 30 mg of powdered imidazole. The negative control eye was treated with 30 μL of saline. After an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

In the first experiment, the test material was determined not to be irritating to the eye. As the test material was solid, the negative results were confirmed by an additional experiment. The second run confirmed the negative results. The positive and negative control substances produced results that were within the historical control data range. The experiment was therefore considered to be valid.

Under the conditions of this study the test material was found to be non irritating to the eye.

Justification for classification or non-classification

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