Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Endpoint summary

Description of key information

Skin:

In an in vitro skin irritation assay (RhE) according to OECD guideline 439, the test item showed skin irritating properties.

In an in vitro skin corrosion assay (RhE) according to OECD guideline 431, the test item did not show skin corrosive properties.

 

Eye:

In an in vitro eye irritation assay (RhCE) according to OECD guideline 492, a mean cell viability of 7.8 % in the test item treated tissues was determined. Thus, the test item is considered to have an effect on the eye (UN GHS Category 1 or 2).

In an ex vivo chicken eye irritation/corrosion assay according to OECD guideline 438, evaluation of the test item resulted in an ICE category classification of 3x I. Thus the test item does not show eye damaging properties.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 April - 23 May, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 201

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT)
- Tissue lot number: 25892
- Date of initiation of testing: 05-04-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure:
3 min: Room temersture
1 h: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After completion of 1 hour period of test item exposure, the tissues were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of PBS) to remove any residual test item/NC/PC.
- 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: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
see: " Any other information on materials and methods"

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered to be “corrosive” to skin in accordance with UN GHS Category 1, if;
Step 1
The tissue viability after 3 minutes exposure is < 50%.
The tissue viability after 3 minutes exposure is ≥ 50% and < 15% after 1 hour exposure.
The test item is considered as non-corrosive to skin in accordance with UN GHS No Category, if the tissue viability after 3 minutes exposure is ≥ 50% and ≥1 5% after 1 hour incubation exposure.
Step 2
A test item identified as being corrosive in step 1 is further subcategorised in accordance with UN GHS based on the following:
The tissue viability after 3 minutes exposure is < 25%: optional subcategory 1A.
The tissue viability after 3 minutes exposure is ≥ 25 %: a combination of optional Sub-Categories 1B-and-1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg + 25 µL water

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min treatment

Value:

96.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h treatment

Value:

79.6

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

Table 2: Summary of the 3 min treatment

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.748	100.0	NC
	±SD	0.046	3.8
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.027	1.6	C (Category 1A)
	±SD	0.003	0.3
	n	2	2
Test Item (Humectol LYS Mod )	Mean	1.656	96.5	NC
	±SD	0.183	15.1
	n	2	2

Table 3: Summary of the 1 h treatment

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.871	100.00	NC
	±SD	0.024	1.8
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.025	1.3	C (Category 1A)
	±SD	0.005	0.4
	n	2	2
Test Item (Humectol LYS Mod )	Mean	1.490	79.6	NC
	±SD	0.268	20.2
	n	2	2

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion assay (RhE) according to OECD guideline 431, the thest item did not show skin corrosive properties.

Executive summary:

The skin corrosive potential of the test item was evaluated in an in vitro skin corrosion assay (RhE) according to OECD guideline 431. In an pre-test the test item did not develope any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Test items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes and 1 h treatment, 50 µL of sterile distilled water (NC) was dispensed into the inserts atop the tissue. Tissues were treated with 25 mg test item + 25 µL distilled water or 50 µL of positive control (glacial acetic acid). Tissues were incubated for 3 min at room temperature or 1 h at 37 °C. At the end of treatment time tissue inserts were rinsed with sterile PBS. In an MTT assay, the viability of the tissues was calculated. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm.

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100 ± 3.8%, 1.6 ± 0.3% or 96.5 ± 15.1%, respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control and clearly represents the irritation potential of positive control. For 1 hour exposure, percentage viability of negative control, positive control and test item was 100 ± 1.8, 1.3 ± 0.4 and 79.6 ± 20.2 respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the laboratory testing conditions, the test item is categorized as non-corrosive to reconstructed human epidermis (RhE) (UN GHS No category), as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 April - 23 May, 2018

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

As recommended in OECD Guideline No. 439, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT) has been selected as test system for in vitro skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT)
- Tissue lot number:
25891
- Date of initiation of testing:
05-04-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure:
35 min at 37 °C (incubator) followed by 25 min at room temperature.
- Temperature of post-treatment incubation (if applicable):
37 °C (incubator)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
The tissues were rinsed with sterile DPBS by filling and emptying the tissue insert for 15 times to remove any residual test item. The constant stream of DPBS was applied from the nearest distance from the tissue surface. After the 15th rinse with washing bottle, the inserts were completely submerged 3 times in approximately 50 mL of DPBS and shaken to remove all traces of test item/NC/PC.
- 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: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
see: " Any other information on materials and methods"

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 item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean tissue viability after exposure and post-treatment incubation is ≤ 50%.
The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

ca. 40 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

11.5

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

Table 1: Individual Tissue optical density (OD) values and viability (%)

Treatment	Tissue No.	Absolute Optical Density (570 nm)		*Blank Corrected Optical Density (nm)		Mean of Blank Corrected Aliquots	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
Negative Control (DPBS)	NC 1	1.909	1.860	1.863	1.814	1.839	101.3
	NC 2	1.861	1.871	1.815	1.825	1.820	100.3
	NC 3	1.841	1.822	1.795	1.776	1.786	98.4
Positive Control (5% Sodium Dodecyl Sulphate)	PC 1	0.109	0.115	0.063	0.069	0.066	3.6
	PC 2	0.114	0.116	0.068	0.070	0.069	3.8
	PC 3	0.114	0.111	0.068	0.065	0.067	3.7
Test Item	TI 1	0.256	0.256	0.210	0.210	0.210	11.6
	TI 2	0.252	0.256	0.206	0.210	0.208	11.5
	TI 3	0.254	0.250	0.208	0.204	0.206	11.4
*Individual Blank Corrected OD values presented after subtracting the mean blank OD value of 0.046 from absolute Optical density values. NC: Negative control, PC: Positive control, TI: Test Item

Interpretation of results:

other: not non-irritating

Conclusions:

In an in vitro skin irritation assay (RhE) according to OECD guideline 439, the test item showed skin irritating properties.

Executive summary:

The skin irritating potential were evaluated in an skin irritation assay (RhE) according to OECD guideline 439. EpiDerm™ (EPI-200-SIT) tissues were topically exposed to 30µLof DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 25 mg of test item. All the treatments were maintained in triplicates. After 60 minutes of exposure with negative control, positive control or test item, the tissues were washed using DPBS. After a post-treatment period of ca. 40 h, cell viability was determined via MTT-Assay. In a pre-test, the test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Mean percentage viability of three replicates of negative control, positive control and test item was 100 ± 1.48%, 3.7 ± 0.09% and 11.5 ± 0.11%, respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the positive control and the suitability of the test method.

Based on the results obtained under the laboratory testing conditions, the test was considered as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

April - May, 2018

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:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: poultry slaughterhouse
- Number of animals: 7
- Characteristics of donor animals: 6 to 7 weeks old and weighing around 1.5 to 2.5 kg
- Storage, temperature and transport conditions of ocular tissue. Heads were removed immediately after sedation of the chickens, by electric shock and incision of the neck for bleeding. The intact heads were transported from the slaughterhouse at ambient temperature (typically between 18 °C and 25 °C) in plastic boxes humidified with tissues moistened with isotonic saline.
- Time interval prior to initiating testing: The procedure involving the collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was completed within two hours to minimize deterioration and/or bacterial contamination.
- indication of any existing defects or lesions in ocular tissue samples: The eyelids of chicken heads were carefully excised, without damaging the cornea. Corneal integrity was quickly assessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds and then rinsed with isotonic saline. Fluorescein – treated eyes were then examined with a slit-lamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).
- 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

Duration of treatment / exposure:

10 s

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids of chicken heads were carefully excised, without damaging the cornea. Corneal integrity was quickly assessed with drop a of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds and then rinsed with isotonic saline. Fluorescein – treated eyes were then examined with a slit-lamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).

EQUILIBRATION AND BASELINE RECORDINGS
After confirming that the eyes were undamaged, the eyes were further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles was cut with a bent, blunt-tipped scissor. All necessary precautions were taken to avoid any corneal damage due to excessive pressure (i.e, compression artifacts). The visible portion of the optic nerve was left attached to the eye when it was removed from the orbit. Immediately after removing the eye from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were removed. The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The entire cornea was supplied with the isotonic saline drip (3 to 4 drops per minute or 0.1 to 0.15 mL/min) by positioning the clamps in the superfusion apparatus. The temperature of the chambers of the superfusion apparatus was maintained at 32 ± 1.5 °C. After being placed in the superfusion apparatus, the eyes were again examined with a slitp-lamp microscope to ensure that there were no damage during the dissection procedure. Corneal thickness was measured at this time using the depth measuring device (Pachymeter). Eyes with (i), a fluorescein retention score of >0.5, (ii) corneal opacity >0.5 or (iii) any additional signs of damage were not selected for the study.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL (NC) USED
Yes, sodium chloride (0.9 % w/v)

POSITIVE CONTROL (PC) USED
Yes, imidazole

APPLICATION DOSE AND EXPOSURE TIME
Test item: 30 mg, 10 s
PC: 30 mg, 10 s
NC: 30 µL, 10s

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

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: isotonic saline (approximately 20 mL) at ambient temperature.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was evaluated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was provided for test item, negative control and positive control group.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was evaluated at the 30 minute observation time point only. The mean fluorescein retention value of all test eyes was calculated for the 30 minute observation time point and used for the overall category score given for test item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Corneal swelling was determined from corneal thickness measurements made with a pachymeter and was expressed as a percentage and is calculated from corneal thickness measurements according to the following formula:

[(Corneal thickness at time t – Corneal thickness at time t = 0) / Corneal thickness at time t = 0] ×100

The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was provided for test item, negative and positive control group.

- Macroscopic morphological damage to the surface: Post treatment, the control and test eyes were evaluated for morphological effects, if any such as “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. The morphological effects were evaluated and recorded individually for all the eyes.

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:

<= 5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Irritation parameter:

cornea opacity score

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

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 7: Data of corneal sweling/thickness and ICE classification

Treatment	Eye No. and Sw%	Corneal Thickness (µm) at t (minutes)
		Selection	0	30	75	120	180	240	ICE Class
Negative Control	1	306	303	288	301	304	298	280	I
	Sw%	NA	NA	-4.95	-0.66	0.33	-1.65	-7.59
Positive control	2	309	297	383	464	487	481	550	IV
	Sw%	NA	NA	28.96	56.23	63.97	61.95	85.19
	3	306	302	377	461	467	486	546
	Sw%	NA	NA	24.83	52.65	54.64	60.93	80.79
	4	305	301	384	468	488	482	544
	Sw%	NA	NA	27.57	55.48	62.13	60.13	80.73
	Mean sw% ±SD	NA	NA	27.12 2.10	54.79 1.89	60.25 4.94	61.00 0.91	82.24 2.55
Test Item (Trial 1)	5	302	294	288	297	301	292	299	I
	Sw%	NA	NA	-2.04	1.02	2.38	-0.68	1.70
	6	298	300	285	299	290	294	297
	Sw%	NA	NA	-5.00	-0.33	-3.33	-2.00	-1.00
	7	298	307	298	285	286	290	284
	Sw%	NA	NA	-2.93	-7.17	-6.84	-5.54	-7.49
	Mean Sw% ±SD	NA	NA	-3.32 1.52	-2.16 4.39	-2.60 4.65	-2.74 2.51	-2.26 4.72
Test Item (Trial 2)	1	302	281	286	286	282	285	292	I
	Sw%	NA	NA	1.78	1.78	0.36	1.42	3.91
	2	276	286	295	292	295	288	285
	Sw%	NA	NA	3.15	2.10	3.15	0.70	-0.35
	3	279	281	287	287	292	281	286
	Sw%	NA	NA	2.14	2.14	3.91	0.00	1.78
	Mean Sw% ±SD	NA	NA	2.35 0.71	2.00 0.20	2.47 1.87	0.71 0.71	1.78 2.13
Sw%: Corneal Swelling percentage, SD: Standard deviation, t: time.

Table 8: Data of corneal opacity and ICE classification

Treatment	Eye No.	Corneal Opacity Scores at t (Minutes)
		Selection	0	30	75	120	180	240	ICE Class
Negative Control	1	0	0	0	0	0	0	0	I
Positive Control	2	0	0	2	3	4	4	4	IV
	3	0	0	2	3	4	4	4
	4	0	0	2	2	4	4	4
	Mean	0	0.0	2.0	2.7	4.0	4.0	4.0
Test Item (Trial 1)	5	0	0	0	0	0	0	0	I
	6	0	0	0	0	0	0	0
	7	0	0	0	0	0	0	0
	Mean	0	0.0	0	0.0	0	0.0	0
Test Item (Trial 2)	1	0	0	0	0	0	0	0	I
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	Mean	0	0.0	0	0.0	0	0.0	0

Table 9: Data of fluorescein retention, morphological effects and ICE classification

Groups	Eye No.	Fluorescein retention at t = (Minutes)			Morphological effects	ICE Class
		Selection	0	30
Negative Control	1	0	0	0	No morphological effects observed	I
Positive Control	2	0	0	1	Loosening of epithelium	III
	3	0	0	1	Loosening of epithelium
	4	0	0	1	Loosening of epithelium
	Mean	0.0	0.0	1.0	NA
Test Item (Trial 1)	5	0	0	0	No morphological effects observed	I
	6	0	0	0	No morphological effects observed
	7	0	0	0	No morphological effects observed
	Mean ±SD	0.0	0.0	0.0	NA
Test Item (Trial 2)	1	0	0	0	No morphological effects observed	I
	2	0	0	0	No morphological effects observed
	3	0	0	0	No morphological effects observed
	Mean ±SD	0.0	0.0	0.0	NA

Interpretation of results:

GHS criteria not met

Conclusions:

In an ex vivo chicken eye irritation/corrosion assay according to OECD guideline 438, evaluation of the test item resulted in an ICE category classification of 3x I. Thus, the test item does not show eye damaging properties.

Executive summary:

The test item was evaluated in the “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” as per OECD Guideline 438. Heads of chickens approximately 6 to 7 weeks old and weighing around 1.5 to 2.5 kg were collected at poultry slaughterhouse. Within 2 hours after killing, enucleated eyes were placed in a susperfusion apparatus. Before dosing, the eyes were incubated for 60 minutes at 32 ± 1.5 °C to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time = 0). Additionally, the fluorescein retention score was also recorded at 0 hour as baseline measurement value. Eyes with corneal opacity <0.5 and fluorescein retention score <0.5 were selected. 30 mg of test item and 30 mg imidazole was applied onto the cornea of three eyes for 10 seconds. Similarly, 30 µL of Sodium Chloride injection IP, 0.9 % w/v was used as negative control and was applied onto the cornea of each eye for 10 seconds. The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescein retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

The in vitro classification for a test item was assessed by reading the GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity and fluorescein retention. The combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 3 x I (ICE class of I observed in all 3 endpoints). This test is considered acceptable as the concurrent negative control and the positive control were identified as GHS Non-Classified and GHS Category 1, respectively.

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphological effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points, the test item was identified for classification as UN GHS No Category.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 April - 07 April, 2018

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)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
As recommended in OECD Guideline for Eye Irritation, EpiOcular™ reconstructed human ocular tissue has been selected as test system for in vitro eye irritation.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ human ocular tissue (OCL-200-EIT) (MatTek Corporation) was used as test system. For further information please refer to "An 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: 50 mg

Duration of treatment / exposure:

5.75 h

Duration of post- treatment incubation (in vitro):

25 min post-soak period
17.75 h Post-treatment incubation

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
Tissue conditioning: 16 h in EpiOcular™ assay medium at standard culture conditions (37±1 °C and 5±1 % CO2).
Pre-treament: Post 16 hours of incubation, the tissues were pre-wetted with 20 μL of DPBS. The plate was tapped to assure that the DPBS spreads the entire tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Exposure: The tissues were incubated at standard culture conditions for 5.75 hours
Rinsing: At the end of treatment time, the test item, negative control and positive control were removed by extensively rinsing the tissues with DPBS.
Post-Soak: After rinsing, the tissues were transferred to and immersed in 5 mL assay medium for 25 minute immersion incubation (Post-Soak) at room temperature.
Post-Incubation: The tissues were incubated for 17.75 hours in CO2 incubator at 37 ± 1 °C and 5 ± 1 % CO2 (Post-treatment incubation).
Tissue viability determination via MMT-assay
- Doses of test chemical and control substances used
50 mg of the test item, 50 µL of negative control or positive control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Expoure: 5.75 h at 37 °C
Post-exposure immersion: 25 min at room temperature
Post-exposure incubation: 17.75 h at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
Test item did not develop colour when dissolved in distilled water and isopropanol. Post incubation optical density of samples were measured at 570 nm and the OD readings were <0.08. Test item was considered as non MTT reducer hence no further additional controls were maintained.
Post incubation, the test item with MTT formed black color. As there was no color change, test item was considered as non-reducer of MTT hence no further additional controls were maintained.
- Number of tissue replicates used per test chemical and controls
2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm, 96-well plate reader
- Description of the method used to quantify MTT formazan
Percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
A test item is labeled as “non-irritant”, if the test item treated percent tissue viability determined by MTT assay is >60% relative to the negative control treated tissue viability.
A test item is labeled as “irritant”, if the percent tissue viability determined by MTT assay is <60% relative to the negative control treated tissue viability.
- Complete supporting information for the specific RhCE tissue construct used
Please refer to "Any other information on materials and methods"

Irritation parameter:

other: % tissue viability

Value:

7.8

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 2: Summary of optical density (OD) and viability (%)

Treatment		OD	Viability (%)	Viability difference between tissues	Classification
Negative Control (Sterile distilled water)	Mean	1.952	100.00	0.92	NI
	±SD	0.036	1.84
	n	2	2	2
Positive Control (Methyl acetate)	Mean	0.041	2.1	0.04	I
	±SD	0.002	0.08
	n	2	2	2
Test Item	Mean	0.153	7.8	0.03	I
	±SD	0.001	0.05
	n	2	2	2
NI = Non Irritant; I = Irritant; n = No. of tissues

Table 3: Individual tissue optical density (OD) values and viability (%)

Treatment	Tissue No.	Absolute Optical Density (570 nm)		*Blank Corrected Optical Density (570 nm)		Mean of Blank Corrected Aliquots	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
Negative Control (Sterile distilled water)	NC 1	2.071	1.962	2.025	1.916	1.970	100.9
	NC 2	1.992	1.969	1.946	1.923	1.934	99.1
Positive Control (Methyl Acetate)	PC 1	0.088	0.088	0.042	0.042	0.042	2.1
	PC 2	0.086	0.087	0.040	0.041	0.040	2.1
Test Item	TI 1	0.201	0.198	0.155	0.152	0.153	7.8
	TI 3	0.199	0.198	0.153	0.152	0.152	7.8
*Individual Blank Corrected OD values presented after subtracting the mean blank OD value of 0.046 from absolute Optical density values. NC: Negative control, PC: Positive control, TI: Test Item

Interpretation of results:

other: not non-irritating

Conclusions:

In an in vitro eye irritation assay (RhCE) according to OECD guideline 492, a mean cell viability of the test item treated tissues was determined. Thus the test item is considered not as non-irritating (UN GHS Category 1 or 2).

Executive summary:

The objective of study was to evaluate the in vitro eye irritation potential of the test item using EpiOcular™ model (OCL-200-EIT) as per the OECD guideline 492. 50 mg of the test item and 50 µL of negative control (sterile deionized water) or positive control (methyl acetate) were dispensed directly atop of EpiOcular™ tissues (Duplicates). The tissues were incubated at standard culture conditions for 5 hours and 45 minutes. At the end of treatment time with test item, negative control and positive control, tissue inserts were rinsed with DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium for 25 minutes immersion incubation (Post-Soak) at room temperature. Thereafter, the tissues were incubated for 17 hours and 45 minutes in CO2 incubator at 37 ± 1 °C and 5 ± 1 % CO2 (Post-treatment Incubation). In a pre-test test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. OD reading was <0.08. Tissue viability was determined via MTT-Assay. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of the tissues was calculated.

Mean percentage viability of negative control, positive control and test item were 100 ± 1.84%, 2.1 ± 0.08% and 7.8 ± 0.05%, respectively. As the mean percentage viability of test item was less than 60% of the negative control considered as irritant, similarly the percentage viability of positive control is less than 60% of negative control clearly represents the irritating potential of PC.

Based on the results obtained and under the laboratory testing conditions, the test item is categorized as irritant (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin:

The skin irritating potential of the test item was evaluated in an skin irritation assay (RhE) according to OECD guideline 439. EpiDerm™ (EPI-200-SIT) tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 25 mg of test item. All the treatments were maintained in triplicates. After 60 minutes of exposure with negative control, positive control or test item, the tissues were washed using DPBS. After a post-treatment period of ca. 40 h, cell viability was determined via MTT-Assay. In a pre-test, the test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Mean percentage viability of three replicates of negative control, positive control and test item was 100 ± 1.48%, 3.7 ± 0.09% and 11.5 ± 0.11%, respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritating potential of the positive control and the suitability of the test method.

Based on the results obtained under the laboratory testing conditions, the test was considered as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control.

Since this assay cannot distinguish between UN GHS category 1 or 2, a follow-up assay was necessary to evaluate the skin corrosive potential of the test item.

The skin corrosive potential of the test item was evaluated in an in vitro skin corrosion assay (RhE) according to OECD guideline 431. In an pre-test the test item did not develope any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Test items were exposed for 1 hour or 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes and 1 h treatment, 50 µL of sterile distilled water (NC) was dispensed into the inserts atop the tissue. Tissues were treated with 25 mg test item + 25 µL distilled water or 50 µL of positive control (glacial acetic acid). Tissues were incubated for 3 min at room temperature or 1 h at 37 °C. At the end of treatment time tissue inserts were rinsed with sterile PBS. In an MTT assay, the viability of the tissues was calculated. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm.

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100 ± 3.8%, 1.6 ± 0.3% or 96.5 ± 15.1% respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control and clearly represents the irritation potential of positive control. For 1 hour exposure, percentage viability of negative control, positive control and test item was 100 ± 1.8, 1.3 ± 0.4 and 79.6 ± 20.2 respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the laboratory testing conditions, the test item is categorized as non-corrosive to reconstructed human epidermis (RhE) (UN GHS No category), as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Conclusion: Since one in vitro test alone cannot predict reliably the skin irritating/corrosive potential of the test item, an integrated test strategy, consisting of the tests according to OECD Guideline 439 (skin irritation) and OECD Guideline 431 (skin corrosion) were performed. According to the results of the performed tests (skin irritating, not skin corrosive) the test item is considered to be skin irritating (UN GHS: Category 2, H315).

Eye:

The objective of study was to evaluate the in vitro eye irritation potential of the test item using EpiOcular™ model (OCL-200-EIT) as per OECD guideline 492. 50 mg each of the test item and 50 µL of negative control (sterile deionized water) or positive control (methyl acetate) were dispensed directly atop of EpiOcular™ tissues (Duplicates). The tissues were incubated at standard culture conditions for 5 hours and 45 minutes. At the end of treatment time with test item, negative control and positive control, tissue inserts were rinsed with DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium for 25 minutes immersion incubation (Post-Soak) at room temperature. Thereafter, the tissues were incubated for 17 hours and 45 minutes in CO2 incubator at 37 ± 1 °C and 5 ± 1 % CO2 (Post-treatment Incubation). In a pre-test the test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. OD reading was <0.08. Tissue viability was determined via MTT-Assay. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of the tissues was calculated.

Mean percentage viability of negative control, positive control and test item were 100 ± 1.84 %, 2.1 ± 0.08 % and 7.8 ± 0.05 %, respectively. As the mean percentage viability of test item was less than 60% of the negative control considered as irritant, similarly the percentage viability of positive control is less than 60% of negative control clearly represents the irritating potential of PC.

Based on the results obtained and under the laboratory testing conditions, the test item is considered as requiring classification and labelling (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

Since this assay cannot distinguish between UN GHS category 1 or 2, a follow-up assay was necessary to evaluated the eye damaging potential of the test item.

The test item was evaluated in the “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” as per OECD Guideline 438. Heads of chickens approximately 6 to 7 weeks old and weighing around 1.5 to 2.5 kg were collected at poultry slaughterhouse. Within 2 hours after killing, enucleated eyes were placed in a susperfusion apparatus. Before dosing, the eyes were incubated for 60 minutes at 32 ± 1.5 °C to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time = 0). Additionally, the fluorescein retention score was also recorded at 0 hour as baseline measurement value. Eyes with corneal opacity <0.5 and fluorescein retention score <0.5 were selected. 30 mg of test item and 30 mg imidazole was applied onto the cornea of three eyes for 10 seconds. Similarly, 30 µL of Sodium Chloride injection IP, 0.9 % w/v was used as negative control and was applied onto the cornea of each eye for 10 seconds. The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescein retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

The in vitro classification for a test item was assessed by reading the GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity and fluorescein retention. The combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 3 x I (ICE class of I observed in all 3 endpoints). This test is considered acceptable as the concurrent negative control and the positive control were identified as GHS Non-Classified and GHS Category 1, respectively.

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphological effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three endpoints, the test item was identified for classification as UN GHS No Category.

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 substance is considered to be classified for skin irritation (UN GHS Category 2, H315) and eye irritation (UN GHS Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.