Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: irritating (OECD 439, GLP, K, rel. 1).

Eye irritation: not irritating (OECD 492, GLP, K, rel.1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02 October 2012 to 08 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 439 and in compliance with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspection date: 10 July 2012/ signed on 30 November 2012)
Specific details on test material used for the study:
- Storage conditions: room temperature in the dark
- Expiration date of the lot/batch: June 2013
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): 12-EKIN-036
- Production date: not reported
- Shipping date: not reported
- Delivery date: 02 October 2012
- Date of initiation of testing: 10 October 2012

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: 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. Remaining DPBS removed with absorbent paper, and if necessary with a cotton-bud.
- 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: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without a reference filter
- Filter bandwidth: NA
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Histology scoring : 21.9 +/- 0.5 (within the specification, i.e. > 19.5)
- Barrier function: IC50 = 2.1 mg/mL (within the specification, i.e. > 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: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous ten months. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3

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

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be 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
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): The test item was used as supplied (undiluted).

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): 5% SDS in distilled water
Duration of treatment / exposure:
15 ± 0.5 min
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:
40.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.8%
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: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 0.644 and the standard deviation value of the percentage viability was 6.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.2%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 2.7%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: Since October 2009 to the commencement of this study (124 studies) the historical mean OD540 of the positive control was 0.059 ± 0.022 and the mean percentage viability was 7.4 ± 3.2. In this same period the historical mean OD540 of the negative control was 0.797 ± 0.086. The positive and negative controls were within the historical ranges for this testing facility.

Table 7.3.1/1: Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540of tissues

Mean OD540of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.630

0.644

0.041

97.8

100*

6.4

0.612

95.0

0.691

107.3

Positive Control Item

0.039

0.044

0.008

6.1

6.8

1.2

0.053

8.2

0.039

6.1

Test Item

0.243

0.262

0.017

37.7

40.7

2.7

0.269

41.8

0.275

42.7

SD=   Standard deviation

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

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test substance is classified for 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 EPISKINTM reconstructed human epidermis model.

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

This test was designed to be compatible with the OECD Guideline No. 429 and method B.46 of Commission Regulation (EC) No. 440/2008/EC and was performed in compliance with GLP.

Triplicate tissues were treated with the test material 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. 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‑labelled 96‑well plate. The optical density was measured at 540 nm.

The relative mean viability of the test item treated tissues was 40.7 ± 2.7 %, after the 15‑minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

With a relative mean tissue viability < 50 %, the test material was considered to be irritant.

Under the experimental conditions of this study, the test substance is classified for 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 (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:
23 to 25 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 492 and in compliance with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: 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) (adopted 28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek in Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava, Slovakia, EpiOcular™ Eye Irritation Test (OCL-200-EIT), Protocol, 14 July 2014
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected from 2015-07-13 to 2015-07-16 / signed on 2015-09-14
Species:
other: human reconstructed cornea model (EpiOcular™)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to A
nimal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to solids, liquids, semi-solids and waxes, so is considered to be applicable to the test item.

- Description of the cell system used:
CELL CULTURE:
- Source: EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA01721, USA).
- Lot No.: 21572
- 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) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ∅).
- Transport: EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate.
- Storage: The day after receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 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 of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (18 hours).
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: deionised water. Positive control: methyl acetate
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
NA
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
duplicate tissues
Details on study design:
- Details of the test procedure used: procedure for liquids
- RhCE tissue construct used, including batch number: EpiOcular™ kits (Lot No.: 21572)
- Doses of test chemical and control substances used: 50 μL
- Duration and temperature of:
exposure: 30 minutes / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
post-exposure immersion: 11-13 min / room temperature
post-exposure incubation period: 120 minutes / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Description of any modifications to the test procedure: none
- Test for direct MTT reduction: 50 μL of the test item are added to 1 mL of a 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 h. Untreated MTT solution was used as a control. Then the colour of the obtained solutions was evaluated.
- Assessment of Color Interference with the MTT endpoint: 50 μL of the test item are added to 2 mL of isopropanol and to 1 mL of water. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour. The isopropanol mixture was left for 3 hours at room temperature.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
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 are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.
Each insert was 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-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight 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. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570). 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
The results are acceptable according to OECD TG 492, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 60% of the negative control viability.
3) 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 killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: not reported
- Complete supporting information for the specific RhCE tissue construct used: yes, attached to the study report
- Reference to historical data of the RhCE tissue construct: no
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes
- Positive and negative control means and acceptance ranges based on historical data: NA
- Acceptable variability between tissue replicates for positive and negative controls: yes
- Acceptable variability between tissue replicates for the test chemical: yes
Irritation parameter:
other: Tissue viability
Run / experiment:
Main study
Value:
93.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.9%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.

OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is > 0.8 and < 2.5 (values between 1.568 and 1.653).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50% of the negative control viability (6.9%).
- Acceptable variability between tissue replicates: yes. The difference of viability between the two relating tissues of a single item is < 20% (values between 0.5% to 7.0%) in the same run (for positive and negative control tissues and tissues of single test items).

Table 7.3.2/1: Results after treatment for 30 minutes

Dose Group

Absorbance (OD) of Tissue 1 and 2


Well 1

Absorbance (OD) of Tissue 1 and 2


Well 2

Mean Absorbance (OD) Tissue 1 and 2

Mean Absorbance (OD)* Tissue 1 and 2 minus Mean Blank

Mean Absorbance (OD) of
2 Tissues*

Rel. Aborbance [%] (Viability)
Tissue 1 and 2**

Absolute value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Rel. Absorbance

[% of Negative Control]** (Viability)

Negative Control

1.596

1.615

1.606

1.568

1.611

97.4

5.3

100.0

1.691

1.691

1.691

1.653

102.6

Positive Control

0.151

0.138

0.144

0.107

0.110

6.6

0.5

6.9

0.147

0.157

0.152

0.114

7.1

Test Item

1.480

1.491

1.485

1.448

1.504

89.9

7.0

93.4

1.459

1.738

1.598

1.561

96.9

Concerning acceptance criteria:

• The negative control OD is > 1.0 and < 2.6 (1.568 and 1.653).

• The mean relative viability of the positive control is below 60% of the negative control viability (6.9%).

•The difference of viability between the two relating tissues of a single item is < 20% (values between 0.5% to 7.0%) in the same run (for positive and negative control tissues and tissues of single test items).

Interpretation of results:
GHS criteria not met
Conclusions:
With a percentage of tissue viability > 60%, the test item does not require classification and labelling according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The study was conducted according to the OECD guideline No. 492 and in compliance with GLP.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also 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 freeze-killed or viable tissues did not have to be performed.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue.

After treatment with the negative control the absorbance values were well within the required acceptability criterion:

• mean OD > 1.0 and < 2.6 (MatTek criterion)

• mean OD > 0.8 and < 2.5 (OECD criterion)

thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 60% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues of a single test item was < 20% in the same run (for positive and negative control tissues and tissues of single test items).

Irritating effects were not observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (93.4%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

With a percentage of tissue viability > 60%, the test item does not require classification and labelling according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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 (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

 

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?

NO

In accordance with the integrated testing strategy, the bottom-up approach normally includes an additional in vitro test for skin corrosion when the result of the in vitro skin irritation test is positive. However, the result of the EpiOcular test being “not an eye irritant”, it can be safely concluded that the substance is also not corrosive to the skin without performing a new test.

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?

YES

 => an Episkin test for irritation was initiated (Bottom-up strategy - substance expected to be non corrosive). Together with the absence of eye irritation in a new in vitro test performed on the substance, the conclusion of this Episkin test is sufficient to conclude on C&L (viability = 40.7 % <=> Skin irritant)

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.

An in vitro skin irritation study was performed (Harlan, 2012). The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

This test was designed to be compatible with the Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 40.7 ± 2.7 %, after the 15‑minute exposure period. With a tissue viability < 50%, the test material was considered to be irritant to skin.

In accordance with the integrated testing strategy, the bottom-up approach normally includes an additional in vitro test for skin corrosion when the result of the in vitro skin irritation test is positive. However, the result of the EpiOcular test being “not an eye irritant”, it can be safely concluded that the substance is also not corrosive to the skin without performing a new test.

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

(at the initiation of the dossier, no test was available)

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

 

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

 => a BCOP assay was initiated: no prediction can be made
=> an EpiOcular assay was initiated: potentially not requiring classification and labelling (mean percent viability > 60%)

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 is submitted at Annex VII level to the REACH regulation

The BCOP assay (Harlan, 2013, Rel.1) was conducted according to the OECD guideline No. 437 and in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The In Vitro Irritancy Score of the test item was 4.0, after the 10 -minute exposure period followed by 120 -minute incubation period. With an IVIS > 3, no prediction can be made as the result is outside the decision criteria (i.e.≤3 or >55).

As a consequence, further in vitro testing was conducted to conclude on eye irritation classification.

The EpiOcular test (Envigo, 2016, Rel.1) was conducted according to the OECD guideline No. 492 and in compliance with GLP.

The quality criteria required for acceptance of results in the test were satisfied. The tissue viability of the test item was 93.4% after the 30 -minute exposure period. With a percentage of tissue viability > 60%, the test item does not require classification for eye irritation.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Skin irritation:

Based on the available information, the substance should be classified as Skin irritant Category 2 (H315: Causes skin irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Eye irritation:

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

Respiratory irritation:

No data was available regarding respiratory irritation.