[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation/corrosion: Skin irritant in vivo (OECD 404, GLP, rel. 1).

Eye irritation: Eye irritant in vivo (OECD 405, GLP, rel.1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 26 to April 23, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 404 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 30-31 January 2017 / Signed on 27 April 2017

Specific details on test material used for the study:

Storage: Fridge, light protected, under nitrogen.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Granja San Bernardo (Tulebras, Navarra - Spain)
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 2.50-2.78 kg
- Housing: Each animal was kept in an individual box installed in conventional air conditioned animal husbanding.
- Diet: foodstuff ((ENVIGO - 2030C), ad libitum
- Water: tap-water from public distribution system, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: at least 10 cycles per hour.
- Photoperiod: 12 hours light/12 hours dark.

IN-LIFE DATES: From 26 March to 23 April 2019

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): Test item was applied as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

Skin reactions were appreciated 1, 24, 48 and 72 hours after removal of the patch and observations were carrried out from Days 7 to 14 (in case of persistent reactions were observed).

Number of animals:

3 males

Details on study design:

PRETEST
Initially, a single animal was treated. After consideration of the cutaneous responses produced in the first treated animal, two additional animals were treated.

TEST SITE
- Area of exposure: Undamaged skin area of the right flank of each animal
- Type of wrap if used: Patch was secured in position with a strip of surgical adhesive tape.

REMOVAL OF TEST SUBSTANCE
- Washing: After removal of the patch, the treated area was rinsed with distilled water.
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
1, 24, 48 and 72 hours after removal of the patch and observations were carrried out from Days 7 to 14 (in case of persistent reactions were observed).

SCORING SYSTEM:
- Method of calculation: Skin irritant reaction was scored as per Draize scale according to OECD guideline 404

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

probability of mild irritation

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

A well-defined to moderate erythema was noted on the treated area 1 hour after the patch removal in all animals and was totally reversible between Days 7 and 14.
A moderate oedema was noted on the treated area 1 and 24 hours after the patch removal in all animals and was totally reversible on Day 7.
On the cutaneous structure, dryness of the skin was noted on Day 7 in all animals. The skin recovered a normal aspect on day 14.

Other effects:

None

Table 7.3.1/1: Individual and mean skin reactions/Erythema - Eschar formation following 4 hour exposure

Skin reaction	Observation time (following patch removal)	Individual Scores - Rabbit Number and Sex
		A7529	A7558	A7559
Erythema/Eschar formation	1 h	2	3	3
	24 h	2	3	3
	48 h	2	3	3
	72 h	2	3	3
	Day 7	1	0	0
	Day 14	0	0	0
	Total (24, 48 and 72 hours)	6	9	9
	Mean (24, 48 and 72 hours)	2.0	3.0	3.0

Table 7.3.1/2: Individual and mean skin reactions/Oedema formation following 4 hour exposure

Skin reaction	Observation time (following patch removal)	Individual Scores - Rabbit Number and Sex
		A7529	A7558	A7559
Oedema formation	1 h	0	3	3
	24 h	3	2	2
	48 h	3	2	2
	72 h	2	2	2
	Day 7	0	0	0
	Day 14	0	0	0
	Total (24, 48 and 72 hours)	8	6	6
	Mean (24, 48 and 72 hours)	2.7	2.0	2.0

Note:

All animals: Dryness on Day 7.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the test conditions, the test material ST 03 C 18 is classified as irritant to the skin (Category 2) according to the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a dermal irritation study performed according to the OECD Guideline No. 404, and in compliance with GLP, 0.5 mL of undiluted test item (ST 03 C 18) was applied on an undamaged skin area of the flank of 3 male New Zealand White rabbits. On the other flank an untreated area was served as the control. Test sites were covered with a semi-occlusive dressing for 4 h. Skin irritation was assessed and scored according to the Draize scale at 1, 24, 48 and 72 hours after removal of the patch and observations were continued from Days 4 to 14. Initially, a single animal was treated. After consideration of the cutaneous responses produced in the first treated animal, two additional animals were treated.

 

A well-defined to moderate erythema was noted on the treated area 1 hour after the patch removal in all animals and was totally reversible between Days 7 and 14.

A moderate oedema was noted on the treated area 1 and 24 hours after the patch removal in all animals and was totally reversible on Day 7.

On the cutaneous structure, dryness of the skin was noted on Day 7 in all animals. The skin recovered a normal aspect on day 14.

 

The mean scores for each animal within 3 scoring times (24, 48 and 72 h) were 2.0 / 3.0 / 3.0 for erythema and 2.7 / 2.0 / 2.0 for oedema. These effects are reversible between days 3 and 7.

Therefore, the test material ST 03 C 18 is classified as irritant to the skin (Category 2) according to the Annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 26 to October 01, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(updated 28 July 2015)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (date of inspection: 21 August 2018 / date of issue: 19 November 2018)

Specific details on test material used for the study:

Storage conditions: Approximately 4 °C in the dark (Note: Store under Nitrogen in a closed container after first opening).

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- EpiSkin™ Tissues (0.38cm2) lot number: 18-EKIN-039
- Maintenance Medium lot number: 18-MAIN3-047
- Assay Medium lot number: 18-ESSC-042
- Production date: not reported
- Shipping date: not reported
- Delivery date: 25 September 2018
- Expiry date: 01 October 2018
- Date of initiation of testing: 26 September 2018

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: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC50 = 2.2 mg/ml (1.5 mg/ml ≤ IC50 ≤ 3.0 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding six months. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: Triplicate water-killed tissues as the test item was found MTT reducer

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

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based upon relative mean tissue viability following the 15 - minute exposure period followed by the 42 - hour post-exposure incubation period
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-Irritant

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 mg/cm2) of the test item was applied to the epidermal surface.
- Concentration (if solution): The test item was used as supplied.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Triplicate tissues for test item, negative and positive controls.
Triplicate water-killed tissues.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

73.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.0

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; the mean OD570 for the negative control treated tissues was 0.761 and the standard deviation value of the viability was 11.2%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes; the relative mean tissue viability for the positive control treated tissues was 7.0% relative to the negative control treated tissues and the standard deviation value of the viability was 2.7%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 15.7%. The test item acceptance criterion was therefore satisfied.
- Historical Control Data Comparison: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding six months.

Table 7.3.1/1: Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.812	0.761	0.085	106.7	100*	11.2
	0.663			87.1
	0.808			106.2
Positive Control Item	0.077	0.053	0.021	10.1	7.0	2.7
	0.039			5.1
	0.044			5.8
Test Item	0.683	0.562	0.119	89.8	73.9	15.7
	0.558			73.3
	0.445			58.5

 

OD=Optical Density

SD= Standard deviation

*= The mean viability of the negative control tissues is set at 100%

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorpored into the testing for correction purpose. However, results of the water-killed tissues was not used for the quantitative correction of results as it was considered unnecessary based on the absence of direct reduction during the main test using the non-viable tissues. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 73.9% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The mean OD₅₇₀ for the negative control treated tissues was 0.761 and the standard deviation value of the viability was 11.2%. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 7.0% relative to the negative control treated tissues and the standard deviation value of the viability was 2.7%. The positive control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 15.7%. The test item acceptance criterion was therefore satisfied.

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 15 to May 20, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 405 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted 09 October 2017

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

TBC_Inspected on / Signed on

Specific details on test material used for the study:

Storage: Fridge, light protected, under nitrogen.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:
- Weight at study initiation: 2.52-2.73 kg
- Housing: Each animal was kept in an individual box installed in conventional air conditioned animal husbanding.
- Diet: foodstuff (SDS - C15), ad libitum
- Water: tap water from public distribution system, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: at least 10 cycles per hour.
- Photoperiod: 12 hours light/12 hours dark.

IN-LIFE DATES: From 15 April to 20 May 2019

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): Test item was instilled, as supplied

Duration of treatment / exposure:

No washing was done

Observation period (in vivo):

Ocular examinations were performed on both right and left eyes 1, 24, 48 and 72 h & Days 7, 14 and 21 following treatment, according to a numerical evaluation.

Number of animals or in vitro replicates:

3 males

Details on study design:

TREATMENT
- 0.1 mL of the test item was instilled, as supplied, into the conjunctival sac of one eye. The other eye remained untreated serving as control.
- Initially, a single animal was treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM: According to OECD guideline 405

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 3 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 3 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 3 days

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 1 day

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 2 days

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 1 day

Irritant / corrosive response data:

The ocular reactions observed during the study have been moderate and totally reversible:
- at the conjunctivae level: a moderate redness noted 1 or 24 hours after the test item instillation in all animals and totally reversible between days 2 and 14, associated with a moderate chemosis noted 1 or 24 hours after the test item instillation in all animals and totally reversible between days 1 and 7;
- at the iris level: an injection was noted 1 hour after the test item instillation in one animal and was totally reversible on Day 3;
- at the corneal level: a moderate opacity, noted 24 hours after the test item instillation in two animals and totally reversible between days 14 and 21.

Other effects:

None

Table 7.3.2/1: Individual and Mean Scores for Cornea, Iris and Conjunctivae

		Cornea	Iris	Conjunctivae
Rabbit Number	Time after Treatment	Corneal Opacity	Iris Lesion	Conjunctival Redness	Conjunctival Chemosis
Animal A7560	1h (D0)	0	1	1	0
	24h (D1)	2	1	2	2
	48h (D2)	2	1	2	2
	72h (D3)	1	0	2	2
	Day 7 (D7)	1	0	1	0
	Day 14 (D14)	1	0	0	0
	Day 21 (D21)	0	0	0	0
	Total (24, 48 and 72h)	5	2	6	6
	Mean (24, 48 and 72h)	1.7	0.7	2.0	2.0
Rabbit Number	Time after Treatment	Corneal Opacity	Iris Lesion	Conjunctival Redness	Conjunctival Chemosis
Animal A7584	1h (D0)	0	0	2	2
	24h (D1)	2	0	1	0
	48h (D2)	1	0	1	0
	72h (D3)	1	0	0	0
	Day 7 (D7)	1	0	0	0
	Day 14 (D14)	0	0	0	0
	Day 21 (D21)	-	-	-	-
	Total (24, 48 and 72h)	4	0	2	0
	Mean (24, 48 and 72h)	1.3	0.0	0.7	0.0
Rabbit Number	Time after Treatment	Corneal Opacity	Iris Lesion	Conjunctival Redness	Conjunctival Chemosis
Animal A7586	1h (D0)	0	0	2	2
	24h (D1)	0	0	1	0
	48h (D2)	0	0	0	0
	72h (D3)	0	0	0	0
	Day 7 (D7)	-	-	-	-
	Day 14 (D14)	-	-	-	-
	Day 21 (D21)	-	-	-	-
	Total (24, 48 and 72h)	0	0	1	0
	Mean (24, 48 and 72h)	0.0	0.0	0.3	0.0

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

Under the test conditions, the test material ST 03 C 18 is classified as irritating to eyes in Category 2 according to the Annex I of the Regulation EC No. 1272/2008 (CLP) and in Category 2A according to the GHS.

Executive summary:

In an eye irritation study conducted according to OECD 405 guideline and in compliance with GLP, 3 New Zealand White male rabbits were exposed to 0.1 mL of test item in one eye while the contralateral eye remained untreated and served as control. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h and Days 7, 14 and 21 following treatment and graded according to the Draize method. Initially, a single animal was treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

 

The ocular reactions observed during the study have been moderate and totally reversible. At the conjunctivae level, a moderate redness noted 1 or 24 hours after the test item instillation in all animals and totally reversible between days 2 and 14, associated with a moderate chemosis noted 1 or 24 hours after the test item instillation in all animals and totally reversible between days 1 and 7. At the iris level, an injection was noted 1 hour after the test item instillation in one animal and was totally reversible on Day 3. At the corneal level, a moderate opacity, noted 24 hours after the test item instillation in two animals and totally reversible between days 14 and 21.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 1.7, 1.0, 0.7 for cornea score; 0.3, 0.0, 0.0 for iris score; 1.7, 1.7, 1.0 for conjunctivae score and 1.3, 1.0, 1.3 for chemosis score.

 

Therefore, the test material, ST 03 C 18, is classified as irritanting to eyes in Category 2 according to the annex I of the Regulation EC No. 1272/2008 (CLP) and in Category 2A according to the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 27 to September 27, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 492 without any deviation.

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:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on July 13-16, 2015/ signed on September 14, 2015

Specific details on test material used for the study:

Storage Conditions: Stored at ~ 4 °C, under nitrogen and protected from light (only valid for storage condition, not for test performance)

Species:

human

Details on test animals or tissues and environmental conditions:

Human cornea model EpiOcular™ tissues
Lot No.: 27069

Cell culture:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts.
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (18 hours).

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): 50 µL

Duration of treatment / exposure:

Treated tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes.

Duration of post- treatment incubation (in vitro):

120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation)

Number of animals or in vitro replicates:

2 tissue replicates each for negative control, test item and positive controls.

Details on study design:

Details of the test procedure used :
- After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 µL (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes.
- At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 12 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
- At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

RhCE tissue construct used, including batch number: Lot No.: 27069

Duration and temperature of exposure:
- Treated tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 30 minutes.

Post-exposure incubation period:
- After rinsing and post-soak immersion, tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

Indication of controls used for direct MTT-reducers and/or colouring test chemicals
- Assessment of direct MTT reduction by the test item: For this purpose approximately 50 μl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item will be presumed to have reduced the MTT.
Since the test item proved to reduce MTT, a functional check using freeze-killed tissue controls (killed controls = KC) had to be performed in at least one definitive assay to evaluate, whether the test material was not binding to the tissue and leading to a false MTT reduction signal.
- Assessment of coloured or staining materials: For this purpose, approximately 50 μL of the test item were added to 1.0 mL of deionised water (glass tube) and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least 1 hour. 1 mL of deionised water was used as control. Additionally 50 μL of the test item were added to 2.0 mL of isopropanol (glass tube) and shaken for 3 hours at room temperature. 2.0 mL of isopropanol was used as control. Two 200 μL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength.
Since the test item did not dye water or isopropanol and the OD of the test item solution was not > 0.08, additional tests with viable tissues did not have to be performed.

Number of tissue replicates used per test chemical and controls (positive control, negative control, test item)
- 2 tissue replicates each for negative control, test item and positive controls.

MTT Assay
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions. Since the test item was colourless inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well. The plates were sealed with parafilm, and were stored over night at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The tissues were pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically with piercing.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
- If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant (no Category according to UN GHS).
- If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

Acceptability of the Assay
The results are acceptable, if:
- the negative control OD is > 0.8 and < 2.5,
- the mean relative viability of the positive control is below 50% of the negative control viability.
- the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
- the positive and negative control data shall fall within the historical control data.

Irritation parameter:

other: Corrected Mean Viability (%)

Run / experiment:

30-minute exposure

Value:

121.07

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

PRE-EXPERIMENT:
-The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
- Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed blue/purple colour indicating that the test item does reduce MTT. Therefore, an additional test with freeze-killed tissues was necessary.

MEAN RELATIVE VIABILITY: The mean absorbance value of the test item, corresponding to the relative cell viability, increased to 121.07% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye. Since the viability value of the test item exposed tissues was 121.07% and hence did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

ACCEPTANCE OF RESULTS:
- The negative control OD is > 0.8 and < 2.5 (2.243 and 2.335).
- The mean relative viability of the positive control is below 50% of the negative control viability (39.03%).
- The difference of viability between the two relating tissues of a single item is < 20% (values between 0.02 p.p and 5.67 p.p) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the killed controls (items and negative control) which were calculated as percent values related to the viability of the relating negative control.

Table 7.3.2/1: Eye irritation in vitro - results

Test Group	Tissue No.	Well 1 [OD570]	Well 2 [OD570]	Mean [OD570] (Well 1 and well 2)	Mean [OD570] blank corr. (Well 1 and well 2)	Mean [OD570] of T1 and T2	Tissue viabil.* [%]	rel. viabil. of T1 and T2**	Diff. of viabil. between T1 and T2 [p.p.]	Mean viability of test item after data correct. procedure [%]
Blank		0.037	0.037	0.037						121.07***
Negative Control	1	2.335	2.250	2.292	2.256	2.240	100.0	100.7	1.39
	2	2.280	2.243	2.261	2.225			99.3
Positive Control	1	0.910	0.886	0.898	0.861	0.874	39.03	38.5	1.15
	2	0.938	0.910	0.924	0.887			39.6
Test Item	1	2.687	2.876	2.781	2.745	2.681	119.68	122.5	5.67
	2	2.671	2.638	2.654	2.618			116.8
Blank		0.037	0.037	0.037
Negative Control Freeze killed Tissues	1	0.100	0.100	0.100	0.063	0.064	2.84	2.8	0.03
	2	0.100	0.101	0.101	0.064			2.9
Test Item Freeze killed Tissues	1	0.069	0.068	0.069	0.032	0.032	1.45	1.4	0.02
	2	0.070	0.069	0.069	0.033			1.5

* Tissue viability = (100 x meanOD of T1&T2_{test item / positive control / negative control}) / meanOD of T1&T2_{negative control}

** Relative Tissue viability = (100 x meanOD blank corrected_{test item / positive control / negative control}) / meanOD of T1&T2_{negative control}

*** Corrected mean viability = Tissue viability_{test item} - (Tissue viability _{KC test item} - Tissue viability _{KC negative control})

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study and with a percentage of tissue viability > 60%, the test item does not require classification for eye irritation or eye damage according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro eye irritation test on the EpiOcular^TM cornea epithelial model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

The test item proved to be an MTT reducer in the MTT pre-test. Its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with viable tissues did not have to be performed, but additional tests with freeze killed tissues were necessary. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes. After rinsing and post-soak immersion, tissues were incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5.0 ± 0.5% CO2 (post-treatment incubation). At the end of the post-treatment incubation, MTT assay was performed and absorbance at 570 nm (OD₅₇₀) was measured to calculate mean relative viability of tissues which was corrected using the freeze-killed tissue viability.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of relative viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). The quality criteria required for acceptance of results in the test were satisfied.

The tissue viability of the test item was 121.07% after the 30-minute exposure period.

Under the experimental conditions of this study and with a percentage of tissue viability > 60%, the test item does not require classification for eye irritation or eye damage according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the skin corrosion/irritation potential of the registered substance:

	Element	Information	Conclusion	Comments
Existing data on physico - chemical properties	1a	Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?	NO
	1b	Is the substance an organic hydroperoxide or an organic peroxide?	NO
	1c	Is the pH of the substance ≤ 2.0 or ≥ 11.5?	NO
	1d	Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?	NO
Existing human data	2	Are there adequate existing human data which provide evidence that the substance is a corrosive or irritant?	NO
Existing animal data from corrosion/irritation studies	3	Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?	NO	(At the initiation of the dossier, no relevant in vivo study was available.)
Existing data from general toxicity studies via the dermal route and from sensitisation studies	4a	Is the substance classified as acutely toxic by the dermal route (Category 1)?	NO
	4b	Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?	NO	(At the initiation of the dossier, no relevant in vivo study was available.)
	4c	Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated exposure?	NO
Existing/new (Q)SAR data and read -across	5a	Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion potential of the substance?	NO
	5b	Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?	NO	(At the initiation of the dossier, no relevant in vivo study was available.)
Existing in vitro data	6a	Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met	NO	(at the initiation of the dossier, no test was available)
	6b	Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	(at the initiation of the dossier, no test was available)
	6c	Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?	NO
Weight-of- Evidence analysis	7	The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?	NO
New in vitro test for corrosivity	8	Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO
New in vitro test for irritation	9	Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	Following the bottom-up approach, an OECD TG 439 (EpiSkin) study was performed. Mean tissue viability = 73,9 % <=> not irritant to skin
New in vivo test for corrosion/irritation	10	Does the substance demonstrate corrosive or irritant properties in an EU/OECD adopted in vivo test? (To be used only as a last resort)	YES	A new OECD TG 404 was performed for other legal requirements (Worldwide notifications). Conclusion = irritant to skin => refutes the results of the in vitro study.

 

An in vitro skin irritation study (Envigo, 2019, Rel.1) was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN reconstructed human epidermis model. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 105.7 %, after the 15‑minute exposure period. Under the test conditions, with a tissue viability > 50%, the test material was considered to be non-irritant to skin.

A new in vivo skin irritation study (Phycher, 2019, Rel.1) was performed according to OECD Guideline 404 for other legal requirements (Worldwide notifications). The mean scores for each animal within 3 scoring times (24, 48 and 72 h) were 2.0 / 3.0 / 3.0 for erythema and 2.7 / 2.0 / 2.0 for oedema, refuting the results of the in vitro study.

In conclusion, the test material was considered to be irritant to skin, based on in vivo study results. The applicablility domain of the in vitro test may be questionned for this category of compound.

Eye irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the eye damage/irritation potential of the registered substance:

	Element	Information	Conclusion	Comments
Conclusion of the information strategy on skin corrosion/irritation	0	Is the substance classified as a skin corrosive?	NO
Existing data on physico - chemical properties	1a	Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?	NO
	1b	Is the substance an organic hydroperoxide or an organic peroxide?	NO
	1c	Is the pH of the substance ≤ 2.0 or ≥ 11.5?	NO
	1d	Are there other physical or chemical properties that indicate that the substance causes serious eye damage or eye irritation?	NO
Existing human data	2	Are there adequate existing human data which provide evidence that the substance has the potential to cause serious eye damage or eye irritation?	NO
Existing animal data from corrosion/irritation studies	3	Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?	NO	(At the initiation of the dossier, no relevant in vivo study was available.)
Existing/new (Q)SAR data and read-across	4	Are there structurally related substances (suitable “read-across” or grouping), which are classified as causing serious eye damage/eye irritation, or indicating that the substance is non-irritant, or do valid (Q)SAR methods indicate serious eye damage/eye irritation or non-irritation of the substance?	NO
Existing in vitro data	5a	Has the substance demonstrated serious eye damage, eye irritation or non-irritating properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	(At the initiation of the dossier, no relevant in vivo study was available.)
	5b	Are there acceptable data from (a) non-validated suitable in vitro test(s), which provide sound evidence that the substance causes serious eye damage/eye irritation?	NO
Weight-of- Evidence analysis	6	The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 0 – 5) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and – if so – how to classify and label?	NO
New in vitro tests for serious eye damage/eye irritation (Annex VII to the REACH Regulation)	7a	Does the substance demonstrate serious eye damage, eye irritation or non-irritant properties in (an) EU/OECD adopted in vitro test(s) for the eye hazard charaterisation? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions of Annex XI are met.	NO	Following the bottom-up approach, an OECD TG 492 (EpiOcular) study was performed. Mean tissue viability = 121,07 % <=> not irritant to skin
	7b	Does the substance demonstrate serious eye damage or eye irritant properties in (a) non-validated suitable in vitro test(s) for serious eye damage/eye irritation?	NO
New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)	8	Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?	YES	A new OECD TG 405 was performed for other legal requirements (Worldwide notifications). Conclusion = irritant to eyes => refutes the results of the in vitro study.

 

An in vitro eye irritation test (Envigo, 2019, rel1) was performed according to the OECD guideline No. 492 and in compliance with GLP, using the EpiOcular test model. The quality criteria required for acceptance of results in the tests were satisfied. The tissue viability of the test item was 121.07% after the 30 -minutes exposure period. Under the test conditions, with a tissue viability > 60%, the test item was considered to be non-irritant to eyes.

A new in vivo eye irritation study (Phycher, 2019, Rel.1) was performed according to OECD Guideline 405 for other legal requirements (Worldwide notifications).The mean scores for each animal within 3 scoring times (24, 48 and 72 h) were 1.7 / 1.3 / 0.0 for opacity; 0.7 / 0.0 / 0.0 for iritis; 2.0 / 0.7 / 0.3 for redness and 2.0 / 0.0 / 0.0 for chemosis, refuting the results of the in vitro study.

In conclusion, the test material was considered to be irritant to eyes, based on in vivo study results. The applicablility domain of the in vitro test may be questionned for this category of compound.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Skin irritation:

Based on the available data, the substance should be classified as as Skin irritant Category 2 according to the CLP and the GHS

Eye irritation:

Based on the available information,the substance should be classified as Eye irritant Category 2 according to the CLP and the GHS.

Respiratory irritation:

No data was available.