Barium(2+) 12-hydroxyoctadecanoate

Endpoint summary

Description of key information

Skin:

In an in vitro skin irritation assay (Episkin) according to OECD guideline 439, a mean tissue viability of 104 % was determined. Therefore the test item was considered to be non-irritant to skin.

Eye:

According to the IATA for eye irritation, one single in vitro/ex vivo assay may not be enough to assess the eye irritating properties of a test item. Therefore, two non in vivo assays were performed (according to OECD guideline 492, ICE and OECD guideline 438, RhCE). In an ex vivo eye irritation assay in chicken eye (ICE) according to OECD guideline 438, an ICE classes combination of 1xI, 2xII was determined. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”. In an in vitro eye irritation assay (RhCE) according to OECD guideline 492, a mean tissue viability of 108.9% was determined. In conclusion, the test item is considered as 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:

from 2017-10-11 to 2017-10-13

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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

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).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM
- Tissue batch number: 17-EKIN-041

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: After the incubation time, the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- 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 MTT
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 ± 10 nm
- Linear OD range of spectrophotometer: Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752)

NUMBER OF REPLICATE TISSUES: 3

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

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

104

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

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

Validity of the test

The mean OD value of the three negative control tissues was 0.897. The mean OD value obtained for the positive control was 0.166 and this result corresponds to 19 % viability when compared to the results obtained from the negative controls. 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.

 

Possible direct MTT reduction with test substance

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 excluded.

Colouring potential of test substances

The test item showed no ability to become coloured in contact with water. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Table 1: Summary of the results

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.874	97
	2	0.853	95
	3	0.965	108
	mean	0.897	100
	standard deviation (SD)		6.62
Positive Control: SDS (5 % aq.)	1	0.136	15
	2	0.181	20
	3	0.181	20
	mean	0.166	19
	standard deviation (SD)		2.95
Test Item:	1	0.971	108
	2	0.892	99
	3	0.931	104
	mean	0.931	104
	standard deviation (SD)		4.40

Table 2: Historical Control Data (Period of 2011-2017 October)

	Negative Control data	Positive Control data
	Phosphate Buffered Saline (1 x PBS)	Sodium Dodecyl Sulphate (SDS) 5 % aq. solution
	Optical Density (OD)	Optical Density (OD)	Viability (% control)
Mean	0.828	0.111	14
Minimum	0.555	0.015	2
Maximum	1.414	0.299	39

Table 3: Quality control of the Episkin SM

Test	Specification	Result
Histology scoring (HES stained vertical paraffin sections)	≥ 19.5	23.4 ± 0.4
IC50 determination (SDS concentration, MTT test)	Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.	Statisfactory
	1.5 mg/mL ≤ IC50 ≤ 3.0 mg/mL	1.9 mg/mL

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro skin irritation assay (Episkin) according to OECD guideline 439, a mean tissue viability of 104 % was determined.

Executive summary:

The skin irritating potential of the test item was assessed in an in vitro skin irritation assay (Episkin) according to OECD guideline 439. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS 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 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

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

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. 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, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 104 %). 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.

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:

from 2018-01-09 to 2018-01-18

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:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The test system was chose according to the OECD guideline 492.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cor¬nea. It consists of highly organized basal cells. These cells are not transformed or trans-fected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm². For further information see "Any other information on materials and methods".

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: ca. 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular™ tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnske Nivy 73, 82105 Bratislava, Slovakia; Batch No: 27019
- Doses of test chemical and control substances used
Test item: ca. 50 mg
positive and negative control: 50 µL each
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
exposure: 6 h
post treatment: 18 h
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: MTT assay via 96-well plate reader

Irritation parameter:

other: % cell viability

Value:

108.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

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 5: Validity of the Experiment

Criterion	Demanded	Found
OD of negative control	> 0.8 and < 2.5	1.7
% mean relative viability of positive control	< 50% of negative control	31.5%
Variation within replicates	< 20%	3.8% (negative control) 6.4% (positive control) 3.4% (test item)

Table 6: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.036	0.038	0.037	0.038	0.037	0.038	0.038	0.039	0.038

Table 7: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Test Item
Tissue 1	1	1.755	0.603	1.779
	2	1.738	0.638	1.893
Tissue 2	1	1.669	0.500	1.886
	2	1.697	0.525	1.900

Table 8: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Test item
Mean - blank (Tissue 1)	1.709	0.583	1.798
Mean - blank (Tissue 2)	1.645	0.475	1.855

Table 9: % Viability Positive Control and Test Item

Designation	Positive Control	Test Item
% Viability (Tissue 1)	34.7%	107.2%
% Viability (Tissue 2)	28.3%	110.6%
% Viability Mean	31.5%	108.9%
standard deviation	4.5%	2.4%

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro eye irritation assay (RhCE) according to OECD guideline 492, a mean tissue viability of 108.9% was determined.

Executive summary:

The eye irritating potential of the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue and a post treatment incubation of 18 h followed. Cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.7. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 31.5 % (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 108.9 %. This value is above the threshold for eye irritation potential (< 60%).

Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcular Eye Irritation Test.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2017-09-13 to 2017-10-05

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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

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
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3-20.8 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. 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).
- Time interval prior to initiating testing: ca 2 h
- indication of any existing defects or lesions in ocular tissue samples: No. 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 saline solution. 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 (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
- 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 µg

Duration of treatment / exposure:

10 s

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180, 240 min

Number of animals or in vitro replicates:

three positive control eyes, three test item treated eyes, one negative control eye

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Selection: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. 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 (i.e., fluorescein retention ≤ 0.5). 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 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. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.

EQUILIBRATION AND BASELINE RECORDINGS
The enucleated 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, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minute. 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 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 saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high 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 the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness, opacity, and fluorescein retention 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. No changes in thickness were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. If an eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES
three positive control eyes, three test item treated eyes, one negative control eye

NEGATIVE CONTROL USED
Yes, NaCl (9 g/L saline) solution

POSITIVE CONTROL USED
Yes, Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 µg, 10 s

OBSERVATION PERIOD
30, 75, 120, 180, 240 min

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 the entire residual test item, if possible


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity
- Damage to epithelium based on fluorescein retention
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm.
- Macroscopic morphological damage to the surface: No

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

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

Irritation parameter:

percent corneal swelling

Value:

4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: up to 240 min, ICE Class I

Irritation parameter:

cornea opacity score

Value:

0.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

fluorescein retention score

Value:

0.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

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 5: Summary of the results

	Corneal thickness (instrument units)								Corneal opacity score						Fluorescein retention score
Relative observation time (min)	-45 - 60	0	Δ (%)	30	75	120	180	240	0	30	75	120	180	240	0	30
N control	60	60	0	60	60	60	60	60	0	0.5	0.5	0.5	0.5	0.5	0	0
	Swelling (%):			0	0	0	0	0	OS:	0.5	0.5	0.5	0.5	0.5	FRS:	0
P control	62	62	0	82	86	88	90	90	0	4	4	4	4	4	0	3
	62	62	0	80	85	86	89	90	0	4	4	4	4	4	0	3
	60	60	0	68	70	70	70	70	0	4	3	4	4	4	0	3
	Mean Swelling (%):			25	31	32	35	36	MOS	3.7	4.0	4.0	4.0	4.0	MFRS:	3.0
Test item	60	60	0	60	61	61	61	61	0	0.5	0.5	0.5	0.5	0.5	0	0.5
	60	60	0	61	62	62	62	62	0	0.5	0.5	0.5	0.5	0.5	0	0.5
	60	60	0	63	64	64	64	64	0	1	1	1	1	1	0	1
	Mean Swelling (%):			2	3	4	4	4	MOS:	0.7	0.7	0.7	0.7	0.7	MFRS:	0.7
Remark: Δ: cornea thickness change between the start of the acclimatization period (t= -45 to -60 min) and the baseline (t=0) MOS: Mean opacity score MFRS: Mean fluorescein retention score P. control: Positive control OS: Opacity score FRS: Fluorescein retention score N. control: Negative control

Interpretation of results:

study cannot be used for classification

Remarks:

Based on the determined ICE classes of 1xII, 2xII, the combination (other combination) resulted in an UN GHS classification of "No prediction can be made".

Conclusions:

In the ex vivo eye irritation assay in chicken eye (ICE) according to OECD guideline 438, an ICE classes combination of 1xI, 2xII was determined. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Executive summary:

The eye irritating potential of the test item was determined in an ex vivo eye irritation assay in isolated chicken eyes (ICE) according to OECD guideline 438. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

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

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

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after 30 and 75 minutes of observation. Furthermore, gentle rinsing with 20 mL saline was performed in all Imidazole and one test item treated eyes after the 120 minutes of observation and all Imidazole treated eyes after the 180 minutes of observation. The cornea surface of Imidazole treated eyes were not totally cleared at 240 minutes after the post-treatment rinse. However, two cornea surfaces out of three test item treated eyes were totally cleared at 120 minutes and all of them was totally (three eyes) cleared at 180 minutes after the post-treatment rinse.

In this ICE, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII.

Positive and negative controls showed the expected results. The experiments were considered to be valid.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin:

The skin irritating potential of the test item was assessed in an in vitro skin irritation assay (Episkin) according to OECD guideline 439. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS 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 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

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

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. 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, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 104 %). 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 (UN GHS No Category).

Eye:

According to the IATA for eye irritation, one single in vitro/ex vivo assay may not be enough to assess the eye irritating properties of a test item. Therefore, two non in vivo assays were performed (according to OECD guideline 492, ICE and OECD guideline 438, RhCE).

The eye irritating potential of the test item was determined in an ex vivo eye irritation assay in isolated chicken eyes (ICE) according to OECD guideline 438. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

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

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

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after the 30 and 75 minutes of observation. Furthermore, gentle rinsing with 20 mL saline was performed in all Imidazole and one test item treated eyes after the 120 minutes of observation and all Imidazole treated eyes after the 180 minutes of observation. The cornea surface of Imidazole treated eyes were not totally cleared at 240 minutes after the post-treatment rinse. However, two cornea surfaces out of three test item treated eyes were totally cleared at 120 minutes and all of them was totally (three eyes) cleared at 180 minutes after the post-treatment rinse.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII.

Positive and negative controls showed the expected results. The experiments were considered to be valid.

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

Since an eye irritating potential of the test item could not be excluded according to the result of the ICE assay, a second assay was performed.

The eye irritating potential of the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue and a post treatment incubation of 18 h followed. Cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.7. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 31.5 % (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 108.9 %. This value is above the threshold for eye irritation potential (< 60%).

Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcular Eye Irritation Test.

In conclusion, the test item is considered as not eye-irritating. This assessment is based on the results of the ex vivo ICE assay (not eye damaging) and the in vitro RhCE assay (not eye-irritating).

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 (EC) No 1272/2008. In vitro results with the test item were negative. As a result the test substance is considered not to be classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.