7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

Endpoint summary

Description of key information

Skin irritation/corrosion: corrosive to the skin (OECD431, GLP, K, rel.1).

Eye damage/irritation: eye damage (classification by default because the substance is corrosive to the skin)

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:

key study

Study period:

05 - 07 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 431 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

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 EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit, MatTek, Bratislava, Slovakia.
- Tissue batch number(s): 23361
- Production Date: 03 October 2016
- Shipping date: 03 October 2016
- Delivery date: 04 October 2016
- Date received: 04 October 2016
- Date of initiation of testing: 06 October 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 and 60 minutes at 37 °C in a humidified atmosphere of 5% CO2
- Temperature of post-treatment incubation: 3 h at 37 °C in a humidified atmosphere of 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was performed using a wash bottle and was achieved by filling and emptying each tissue approximately 20 times under a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test item.
- Observable damage in the tissue due to washing: no noted damage to the test item tissue culture surfaces following the rinsing step.
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: not reported
- Filter bandwidth: filter band pass ± 15
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.
- Viability: OD (540-570 nm) = 1.739 ± 0.134 (acceptance criteria: between 1.0-3.0)
- Barrier function: ET-50 = 6.71 hours (acceptance criteria: between 4.77-8.72 hours)
- Morphology: Tissue viability and the barrier function test are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile (Bacteria, yeast and other fungi not detected)
- Reproducibility: For the previous 39 experiments conducted between April 2016 and September 2016 using this test method, the mean OD of the positive control was 0.087 ± 0.022 after 3 minutes of exposure and 0.078 ± 0.023 after 60 minutes of exposure. The mean percentage viability was 4.7 ± 0.9 after 3 minutes of exposure and 4.4 ± 0.8 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤15% relative to the negative control treated tissues after 1 hour exposure). In this same period the mean OD of the negative control was 1.922 ± 0.239 after 3 minutes of exposure and 1.866 ± 0.238 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was ≥0.8 and ≤2.8 for every exposure time).

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

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues : Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : 2
- Method of calculation used: True viability = mean OD (treated viable tissues) - [OD (treated killed tissues)-OD (untreated killed tissues)]

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water was used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied

Duration of treatment / exposure:

3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Duplicate tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5.5%

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

8.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5.5%

Remarks on result:

other: Positive indication of corrosion

Other effects / acceptance of results:

TISSUE VIABILITY:
The relative mean viability of the test item treated tissues was 103.7 and 8.2% after 3 and 60 minutes exposure, respectively.
The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure.
The relative mean viability of the positive control treated tissues was 5.5 after 3 and 60 minutes exposure.

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT. Direct reduction was <30% relative to the negative control and therefore acceptable.
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.709 for the 3 Minute exposure period and 1.742 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Historical data: In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.

Table 7.3.1/1: Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562of duplicate tissues (tvt)	Corrected OD562 of tissues (tvt-[tkt-ukt])	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.625	1.709	-	0.119	7.0	100*
		1.793
	60 Minutes	1.774	1.742	-	0.046	2.6
		1.709
Positive Control	3 Minutes	0.099	0.094	-	0.008	N/A	5.5
		0.088
	60 Minutes	0.099	0.096	-	0.005	N/A	5.5
		0.092
Test Item	3 Minutes	1.733	1.776	1.772	0.060	3.4	103.7
		1.818
	60 Minutes	0.254	0.269	0.144	0.021	7.6	8.2
		0.283

tvt = treated viable tissues

tkt = treated killed tissues

ukt = untreated killed tissues

3 minute exposure corrected mean OD₅₆₂:

x̅ (0.157 + 0.156) = 0.157 (tkt) – x̅ (0.159 + 0.147) = 0.153 (ukt) = 0.004

60 minute exposure corrected mean OD562:

x̅ (0.319 + 0.255) = 0.287(tkt) – x̅ (0.174 + 0.149) = 0.162 (ukt) = 0.125

3 minute exposure direct MTT reduction relative to the negative control = 0.2%
60 minute exposure direct MTT reduction relative to the negative control = 7.2%

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

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Remarks:

combination of category 1B-and-1C

Conclusions:

Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

Duplicate tissues were treated with 25 mg of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact for exposure periods of 3 and 60 minutes. The test item was found to directly reduce MTT. Direct reduction was <30% relative to the negative control and therefore acceptable. Thus additional non-viable tissues were incorporated into the testing for correction purposes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 562 nm (OD₅₆₂). 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 103.7 and 8.2% after 3 and 60 minutes exposure, respectively. The relative mean viability of the positive control treated tissues was 5.5% after 3 and 60 minutes exposure. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 December 2015 to 8 February 2016

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:

adopted 28 July 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on November 25 to 27, 2015 / Signed on February 15, 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, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 15 EKIN 050 (test 1) and 16 EKIN 005 (test 2)
- Production date: not reported
- Shipping date: 15 December 2015 (test 1) and 2 February 2016 (test 2)
- Delivery date: 15 December 2015 (test 1) and 2 February 2016 (test 2)
- Expiry date: 23 December 2015 (test 1) and 8 February 2016 (test 2)
- Date of initiation of testing: 15 December 2015 (test 1) and 2 February 2016 (test 2)

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the treatment period, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove any residual test or control items and then incubated in 2mL maintenance medium for 42 h at 37 °C, 5 % CO2 in air. Number of washing steps not reported.
- 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: BMG Fluostar Optima – plate reader
- Wavelength: 540 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.722, 0.732 and 0.702 (mean historical OD of the negative control was 0.854 ± 0.148)
- Barrier function: IC50 = 2.3 mg/ml (test 1) and 2.1 mg/ml (test 2) ( ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: For the previous 68 experiments conducted between October 2008 and November 2015 using this test method, the mean OD of the positive control was 0.160 ± 0.074 and the mean percentage viability was 20.8 ± 9.3 (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18%). In this same period the mean OD of the negative control was 0.782 ± 0.087 (The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was ≥0.6 and ≤1.5).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2. The initial test was conducted between 15 December 2015 and 21 December 2015, however, the mean absorbance of the triplicate negative control values was 0.551 which was below the minimum acceptance value of 0.6. The test was repeated between 2 February 2016 and 8 February 2016. The data and results from these two tests are included in the report.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg of the test substance (powder) was dispensed over each tissue. The tissues were wetted with 5 μL of purified water prior to application of the test substance.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): DPBS

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 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues for test substance, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

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

Value:

28.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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: The mean absorbance of the triplicate negative control values for the original test (test 1) was 0.551 which was below the minimum acceptance value of 0.6. The mean absorbance for the negative control in the repeat test (test 2) was 0.746 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability for test 1 and 2 was 8.3 and 6.1 respectively. Both test values were below the maximum value of 18. The negative control acceptance criteria were therefore met for the test 2.
- Acceptance criteria met for positive control: The percentage mean viability of the positive control for test 1 was 17.3 ± 0.3 and test 2 was 10.3 ± 3.7 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%. The positive control acceptance criteria were therefore met.
- Acceptance criteria met for variability between replicate measurements: Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with test item compared to the negative control tissues was 28.6% in test 2. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues were 18.2%, which was above the acceptance criterion of 18.0. In test 1, the standard deviation calculated from individual tissue viabilities was 25.9. The results from test 2 are consistent with the first test in that both % viabilities are less than 50% and the standard deviations are greater than 18, which may be possibly due to test material sticking to and/or penetrating the Episkin tissues. As the standard deviation for the repeat test was only 0.2 greater than 18.0 and that all other acceptance criteria were met the repeat test was considered valid.
- Range of historical values if different from the ones specified in the test guideline: For the previous 68 experiments conducted between October 2008 and November 2015 using this test method, the mean OD of the positive control was 0.160 ± 0.074 and the mean percentage viability was 20.8 ± 9.3. In this same period the mean OD of the negative control was 0.782 ± 0.087.

Other effects:

None

Table 7.3.1/1: EpiSkin™ results

Test	Sample	Tissue viability as % of mean OD negative control				Prediction MTT endpoint
		Replicate Tissues			Mean ± SD
		a	b	c
Test 1	Test substance	39.0	9.6	61.3	36.7 ± 25.9	Category 2
	Negative control	97.6	93.2	109.20	100.0 ± 8.3	Not applicable
	Positive control	17.4	17.4	16.9	17.3 ± 0.3	Category 2
Test 2	Test substance	39.1	39.0	7.6	28.6 ± 18.2	Category 2
	Negative control	99.7	94.0	106.3	100.0 ± 6.1	Not applicable
	Positive control	13.9	6.5	10.5	10.3 ± 3.7	Category 2

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test substance is classified as H315 “Causes Skin Irritation” Category 2 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 10 ± 2 mg of the test substance for an exposure period of 15 ± 0.5 minutes. The tissues were wetted with 5 μL of purified water prior to application of the test substance. At the end of the exposure period each tissue was rinsed before incubating for 42 h. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a biopsy of each epidermis was made and the tissues were placed into acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period, duplicate 200 μL samples were transferred to the wells of 96-well plate and the optical density was measured at 540 nm.

 

The test substance was tested on two separate occasions. In the initial test (test 1), the mean absorbance of the triplicate negative control values was 0.551 which was below the minimum acceptance value of 0.6. As test 1 was considered invalid the test was repeated. In the repeat test (test 2), negative and positive controls results were within the acceptable range and the test was considered valid and the quality criteria required for acceptance of results in the test were satisfied. The conclusion was based on the results from the repeat test (test 2).

 

The test substance elicited a mean tissue viability of 28.6 ± 18.2% in test 2. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues were 18.2%, which was above the acceptance criterion of 18.0. In test 1, the standard deviation calculated from individual tissue viabilities was 25.9. The results from test 2 are consistent with the first test in that both % viabilities are less than 50% and the standard deviations are greater than 18, which may be possibly due to test material sticking to and/or penetrating the Episkin tissues. As the standard deviation for the repeat test was only 0.2 greater than 18.0 and that all other acceptance criteria were met the repeat test was considered valid.

 

Under the experimental conditions of this study, the test substance is classified as H315 “Causes Skin Irritation” Category 2 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:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) 1907/2006 as amended: Annex VII, section 8.2: the serious eye damage / eye irritation study does not need to be conducted if the substance is classified as skin corrosion, leading to classificiation as serious eye damage (Category 1). The substance was shown to be corrosive to the skin in a new in vitro skin corrosion EpiDerm test (Envigo, 2017, Rel.1), therefore the substance was considered to be able to induce serious eye damage and no further in vitro test was needed.

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion:

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 contact with air (pyrophoric) or water 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
Existing data from general toxicity studies via the dermal route and from sensitisation studies	4a	Is the substance classified as fatal in contact with skin (LD50 ≤ 50 mg/kg bw, CLP hazard statement H310)	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	LLNA available: tested only up to 30%. No sign of irritation or corrosion leading ot C&L. NB: In general, irritation data from the Local Lymph Node Assay are not usable. The test substance is applied to the dorsum of the ear by open topical application No local effects were reported in acute oral study at 2000 mg/kg bw (in corn oil)
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
	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 irritation	8	Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?	YES	=> an Episkin test for irritation was initiated (Bottom-up strategy - substance expected to be non corrosive). The conclusion of this Episkin test is not sufficient to conclude on C&L, a skin corrosion test was performed to assess the potential corrosivity of the substance to the skin.
New in vitro test for corrosivity	9	Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?	YES	=> an EpiDerm test for corrosion was initiated (Bottom-up strategy - substance expected to be non corrosive). The conclusion of this EpiDerm test is sufficient to conclude on C&L (3 minutes = 103.7%; 60 minutes = 8.2% => corrosive category 1B/1C)
New in vivo test for corrosion/irritation	10	To be used only as a last resort	NO	In vivo testing should not be conducted in this case since the substance falls under the scope of the specific in vitro tests performed, and there are no substance-specific limitations on use of those tests. An adaptation according to Annex XI to the REACH Regulation is included in this dossier.

Following the REACH bottom-up strategy, an in vitro skin irritation study (Envigo, 2016, Rel.1) was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN reconstructed human epidermis model. The test substance was tested on two separate occasions. In the initial test (test 1), the mean absorbance of the triplicate negative control values was 0.551 which was below the minimum acceptance value of 0.6. As test 1 was considered invalid the test was repeated. In the repeat test (test 2), negative and positive controls results were within the acceptable range and the test was considered valid. The standard deviation calculated from individual tissue viabilities of the three identically test substance treated tissues were 18.2%, which was above the acceptance criterion of 18.0. In test 1, the standard deviation calculated from individual tissue viabilities was 25.9. The results from test 2 are consistent with the first test in that both % viabilities are less than 50% and the standard deviations are greater than 18, which may be possibly due to test material sticking to and/or penetrating the Episkin tissues. As the standard deviation for the repeat test was only 0.2 greater than 18.0 and that all other acceptance criteria were met the repeat test was considered valid. The conclusion was based on the results from the repeat test (test 2). The relative mean viability of the test item treated tissues was 28.6 %, after the 15‑minute exposure period. With a tissue viability < 50%, the test material was considered to be irritant to skin.

In compliance with bottom-up strategy and to determine the potential of the substance to induce skin corrosion, an in vitro skin corrosion study (Envigo, 2017, Rel.1) was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model. The relative mean viability of the test item treated tissues was 103.7 and 8.2% after 3 and 60 minutes exposure, respectively. The relative mean viability of the positive control treated tissues was 5.5% after 3 and 60 minutes exposure. The quality criteria required for acceptance of results in the test were satisfied. With a tissue viability < 15% after 60 minutes of exposure, the test material was considered to be corrosive to skin.

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 contact with air (pyrophoric) or water 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
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 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	The substance was shown to be corrosive to the skin in a new in vitro skin corrosion EpiDerm test => Therefore the substance was considered to be able to induce serious eye damage and no further in vitro test was needed as the conclusion of this skin corrosion test is sufficient to conclude on C&L
	8b	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)	8b	Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?	NO	In vivo testing should not be conducted in this case since the substance falls under the scope of the specific in vitro tests performed, and there are no substance-specific limitations on use of those tests. An adaptation according to Annex XI to the REACH Regulation is included in this dossier.

Since the substance was shown to be corrosive to the skin in the new in vitro skin corrosion EpiDerm test, therefore the substance was considered to be able to induce serious eye damage and no further in vitro test was needed as the conclusion of this skin corrosion test is sufficient to conclude on C&L.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available information, the substance should be classified as skin corrosive sub-category 1B (H314 “Causes severe skin burns and eye damage) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Therefore, the substance is also classified for serious eye damage Category 1 according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available regarding respiratory irritation. In general, a classification for corrosivity is considered to implicitly cover the potential to cause respiratory tract irritation and so the additional Category 3 is considered to be superfluous. However, such substance has to be supplementary labelled with EUH071 if there is a possibility of exposure via inhalation taking into consideration the saturated vapour concentration and the possibility of exposure to particles or droplets of inhalable size. It is also strongly recommended to apply the precautionary statement P260: "Do not breathe dust/fume/gas/mist/vapours/spray."