2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Endpoint summary

Description of key information

Skin irritation/corrosion: Not irritating (OECD 439, GLP, K, rel. 1).

Eye irritation: Severe eye damage, top-down approach: ICE: Category 1 (irreversible effects on the eye) (OECD 438, GLP, WoE, rel.1). However, test item adherence was noted in 2/3 treated eyes and this could have influenced the results, leading to an overestimated classification. An other in vitro test is ongoing (EpiOcular, OECD TG 492) to confirm or dispute the results.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October to 06 November 2017

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)

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 (inspected on 05 July 2016/ signed on 28 October 2016)

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: 17-EKIN-044
- Maintenance Medium lot number: 17-MAIN3-046
- Assay Medium lot number: 17-ESSC-043
- Production date: not reported
- Shipping date: not reported
- Delivery date: 31 October 2017
- Expiry date: 06 November 2017
- Date of initiation of testing: 31 October 2017

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 = 2.0 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: Not a 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): Approximately 10 mg (26.3 mg/cm2) of the test item was applied to the epidermal surface. 5 µL of sterile distilled water was topically applied previously to the epidermal surface in order to improve contact between the test item and the epidermis.
- 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

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

Value:

105.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

7.2

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 0.844 and the standard deviation value of the viability was 7.8%. 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 7.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.5%. 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 6.9%. 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	0.914	0.844	0.066	108.3	100*	7.8
	0.784			92.9
	0.834			98.8
Positive Control Item	0.072	0.061	0.021	8.5	7.2	2.5
	0.074			8.8
	0.037			4.4
Test Item	0.831	0.892	0.058	98.5	105.7	6.9
	0.947			112.2
	0.899			106.5

 

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. 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 105.7% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The mean OD₅₇₀ for the negative control treated tissues was 0.844 and the standard deviation value of the viability was 7.8%. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 7.2% relative to the negative control treated tissues and the standard deviation value of the viability was 2.5%. 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 6.9%. 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.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September to 31 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 438 without any deviation.

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK.
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day (4th September 2017).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. All eyes were placed in the superfusion chamber by 9.36 am (4th September 2017). Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None

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): 0.03 g of test item (undiluted) was applied to the cornea
- For the purpose of this study the test item was used as supplied.

Duration of treatment / exposure:

The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline

Duration of post- treatment incubation (in vitro):

Evaluation of the corneas at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes have been decontaminated

Number of animals or in vitro replicates:

Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
- Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
- Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32.0 ± 1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.
- Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED
0.03 mL of the negative control item, Sodium chloride 0.9% w/v, was applied to the cornea of each negative control eye.

POSITIVE CONTROL USED
0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye.

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish.
- 0.03 g of test item was applied to the cornea and the test item remained in place for 10 seconds.

OBSERVATION PERIOD
- Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

METHODS FOR MEASURED ENDPOINTS:
- Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
- Macroscopic morphological damage to the surface: Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
[(corneal thickness at time (t) / corneal thickness at time = 0) / corneal thickness at time =0] x 100
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

10 seconds exposure

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class IV

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

10 seconds exposure

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class IV

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

10 seconds exposure

Value:

26.64

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class IIII

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: yes, corneal opacity. No morphological effects were noted in the test item or control item treated eyes. Test item adherence was noted in 2/3 treated eyes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
Acceptance criteria met for negative control: Yes; Maximal mean score for corneal opacity: 0.5; Mean score of Fluorescein Retention: 0.5; Maximal mean corneal swelling compared to time zero: 2.86%
Acceptance criteria met for positive control: Yes; Maximal mean score for corneal opacity: 4.0; Mean score of Fluorescein Retention: 3.0; Maximal mean corneal swelling compared to time zero: 50.48%
In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory. The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

Table 7.3.2/3: Individual Scores and Mean Scores for Corneal Effects – Test Item

End Point	Eye Number	Time (after decontamination)
		0 minutes	30 minutes	75 minutes	120 minutes	180 minutes	240 minutes
Corneal Opacity	3B	0.5	4	4	4	4	4
	6B	0.5	2	2	2	2	2
	8B	0	2	2	2	3	3
Mean		0.3	2.7	2.7	2.7	3.0	3.0
ICE Class		IV
Fluorescein Retention	3B		2
	6B		1
	8B		3
Mean			2.0
ICE Class		IV
Corneal Thickness	3B	0.72	0.82	0.96	0.96	0.96	0.96
	6B	0.70	0.77	0.82	0.82	0.80	0.84
	8B	0.72	0.86	0.86	0.93	0.84	1.02
Mean		0.71	0.82	0.88	0.90	0.92	0.90
Mean Corneal Swelling (%)			14.49	23.36	26.64	25.23	26.17
ICE Class		III
Corneal Epithelium Condition	3B		TA	TA	TA	TA	TA
	6B		N	N	N	N	N
	8B		TA	TA	TA	TA	TA
ICE Classes Combined:		2 x IV; 1 x III
Classification:		Category 1

N= Normal

TA = Test item adherance

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the test conditions, the test item is classified as Category 1 (irreversible effects on the eye) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 

The test item was applied, as supplied, at 0.03 g onto the cornea of each of three enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 3.0, corresponding to the ICE class IV;

- mean score of fluorescein retention: 2.0, corresponding to the ICE class IV;

- maximal mean corneal swelling: 26.64% at 120 min after treatment corresponding to the ICE class III.

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, Imidazole, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Under the test conditions, the test item is classified as Category 1 (irreversible effects on the eye) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Test item adherence was noted in 2/3 treated eyes and this could have influenced the results, leading to an overestimated classification. An other in vitro test is ongoing (EpiOcular, OECD TG 492) to confirm or dispute the results.

Endpoint conclusion

Endpoint conclusion:

no study available

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 (December 2016), 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 test 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
	4c	Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated exposure?	NO	(at the initiation of the dossier, no test was available)
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
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.	YES	Following the bottom-up approach, an OECD TG 439 (EpiSkin) study was performed. Mean tissue viability = 105.7 % <=> not irritant to skin
New in vivo test for corrosion/irritation	10	To be used only as a last resort	NO

The in vitro skin irritation study (Envigo, 2017, 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. With a tissue viability > 50%, the test material was considered to be non-irritant to skin.

Moreover, the test material was not predicted to be phototoxic using the 3T3 NRU Phototoxicity Assay according to the OECD Guideline No. 432 and the EURL ECVAM DB-ALM (INVITTOX) Protocol No. 78 (Ceetox, 2013), up to 100µg/ml (highest allowable concentration).

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 (December 2016), 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	No alert using Toxtree (v2.6.6)
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 test 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.	YES	An in vitro test for corrosion (ICE, OECD TG 438) was performed. Combination of the three endpoints is 2 x IV, 1 x III <=> Corrosive to the eyes. Test item adherence was noted in 2/3 treated eyes and this could have influenced the results, leading to an overestimated classification.
	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?		An other in vitro test is ongoing at the time of dossier submission (EpiOcular, OECD TG 492) to confirm or dispute the results.
New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)	8b	Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?	NO

 

The ICE test (Envigo, 2017, Rel.1) was conducted according to the OECD guideline No. 438 and in compliance with GLP. The ocular reactions observed in eyes treated with the test item were: maximal mean score of corneal opacity: 3.0, corresponding to the ICE class IV; mean score of fluorescein retention: 2.0, corresponding to the ICE class IV; maximal mean corneal swelling: 26.64% at 120 minute corresponding to the ICE class IV. The combination of the three endpoints for the test item was 2 x IV, 1 x III. The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, Imidazole, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Test item adherence was noted in 2/3 treated eyes and this could have influenced the results, leading to an overestimated classification. An other in vitro test is ongoing (EpiOcular, OECD TG 492) to confirm or dispute the results.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

No additional self-classification is proposed regarding skin irritation according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Based on the available information, the substance should be classified as eye damage Category 1 (H318: "Causes serious eye damage) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS. However, test item adherence was noted in 2/3 treated eyes and this could have influenced the results, leading to an overestimated classification. An other in vitro test is ongoing (EpiOcular, OECD TG 492) to confirm or dispute the results.

No data was available regarding respiratory irritation.