Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin corrosion / irritation: Standard quality (Item 2) is not corrosive (OECD431, GLP, K, rel.2). Standard quality (Items 1, and 4) and high alpha pinene quality (item 5) are not irritant (OECD 439, GLP, K, rel.1)

Eye damage/irritation: Standard quality (Item 2) is not irritant (similar to OECD 437, GLP, K, rel.2)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-28 April 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD test Guideline No. 431. However, functional model conditions and references to historical control data are not included in the report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted April 13, 2004
Deviations:
yes
Remarks:
functional model conditions and references to historical control data are not included in the report.
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
yes
Remarks:
functional model conditions and references to historical control data are not included in the report.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
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: Epi-200-Kit, MatTek Corporation (Ashland, MA 01721, USA).
- Lot number: 10504 Kit N
- Production Date: no data
- Shipping date: no data
- Delivery date: no data
- Date received: April 24, 2008
- Date of initiation of testing: April 25, 2008

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: At the end of the exposure period the first insert was removed from the 6-well plate. Using a wash bottle the tissue was gently rinsed with PBS Rinse Solution to remove any residual test material. Excess PBS Rinse Solution was removed by gently shaking the insert and
blotting the lower surface with blotting paper.
- 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: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Versamax® Molecular Devices, D-85737 Ismaning
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Not reported

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

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not needed

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): 50 µL of the undiluted test item.

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:
92.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
26.4%
Remarks on result:
other: No indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
85.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
12.4%
Remarks on result:
other: No indication of corrosion
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not included in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.637 for the 3 Minute exposure period and 1.703 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied (≥ 0.8).
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 26.4% and 12.4% relative to the negative control following the 3 and 60 Minute exposure period, respectively. The positive control acceptance criterion was therefore satisfied (≤ 30%).

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


Dose group

Treatment

Interval

Absorbance

570 nm

Tissue 1*

Absorbance

570 nm

Tissue 2*

Mean

Absorbance

of 2 Tissues

Rel. Absorbance

[% of Negative

Control]**

Negative Control

3 min

1.628

1.647

1.637

100.0

 

Positive Control

3 min

0.494

0.369

0.432

26.4

Pink Pepper (ST

08 C 08)

3 min

1.547

1.486

1.517

92.6

 

Negative Control

1 hour

1.755

1.650

1.703

100.0

 

Positive Control

1 hour

0.191

0.231

0.211

12.4

 

Pink Pepper (ST

08 C 08)

1 hour

1.436

1.477

1.456

85.5

 

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: 100 x (absorbance test item) / (absorbance negative control)

Interpretation of results:
other: Non-corrosive base don GHS criteria
Conclusions:
Under the experimental conditions of this study, the test substance is not classified as corrosive, 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 50 µL of the undiluted test item for exposure periods of 3 and 60 minutes. 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 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 92.6% and 85.5% after 3 and 60 minutes exposure, respectively. The relative mean viability of the positive control treated tissues was 26.4% and 12.4% 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 not classified as corrosive 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 corrosion endpoint.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-29 May 2017
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
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on July 05, 2016 / Signed on October 28, 2016)
Specific details on test material used for the study:
Storage conditions: Room temperature in the dark until 19 January 2017 thereafter approximately 4°C in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH top-down 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): 17-EKIN-019
- Production date: not reported
- Shipping date: not reported
- Delivery date: 10 May 2017
- Expiry date: 15 May 2017
- Date of initiation of testing: 11 May 2017

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 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each wellsidual 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:
Deviation 1
Due to unusual variation noted in the untreated killed control group, it was considered necessary to repeat the killed tissue groups.
Deviation 2
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

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 = 1.8 mg/ml ( ≥ 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 six months. This was taken to show the correct functioning of the test system.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Fresh tissues / killed tissues: skin irritation potential performed in parallel on viable and water killed tissues for quantitative correction of the results
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO2 in air for 48 +/- 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to +30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
- N. of replicates : 3 (test item + untreated)
- Method of calculation used: the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

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

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 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:
95.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
12.2%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: yes, an assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.656 and the standard deviation value of the viability was 7.3%.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%.
- 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 11.5%.
- Reference to historical values: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

Table 7.3.1/1: EpiSkin™ results

Item

OD570of tissues

Mean OD570of triplicate tissues

±SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.661

0.656

0.048

100.8

100*

7.3

0.701

106.9

0.606

92.4

Positive Control Item

0.084

0.080

0.005

12.8

12.2

0.7

0.081

12.3

0.075

11.4

Test Item

0.549

0.629

0.076

83.7

95.9

11.5

0.640

97.6

0.699

106.6


OD=Optical Densit

SD=       Standard deviatio

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

Interpretation of results:
other: CLP 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).
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.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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.

 

The relative mean viability of the test item treated tissues was 95.9% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 12.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.656 and the standard deviation value of the viability was 7.3%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.5%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

 

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

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 30 to February 4, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 439.
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
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on August 21, 2018 / Signed on November 19, 2018)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH top-down 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): 19-EKIN-005
- Production date: not reported
- Shipping date: not reported
- Delivery date: 29 January 2019
- Expiry date: 4 February 2019
- Date of initiation of testing: 30 January 2019

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 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each wellsidual 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:
Deviation 1
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

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 = 1.9 mg/ml ( ≥ 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
absence of HIV1 & 2 & hepatitis C antibodies; absence of hepatitis B antigen HBs
- 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 six months. This was taken to show the correct functioning of the test system.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

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

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 µL

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 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:
96.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
3.9%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: yes, an assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.846 and the standard deviation value of the viability was 6.8%. 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 12.2% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%.
- 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 13.8%. The test item acceptance criterion was therefore satisfied.

Table 7.3.1/1: EpiSkin™ results

Item

OD570of tissues

Mean OD570of triplicate tissues

±SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.814

0.846

0.058

96.2

100*

6.8

0.812

96.0

0.913

107.9

Positive Control Item

0.046

0.033

0.011

5.4

3.9

1.3

0.029

3.4

0.025

3.0

Test Item

0.937

0.813

0.117

110.8

96.1

13.8

0.705

83.3

0.797

94.2


OD=Optical Density

SD=       Standard deviation

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

Interpretation of results:
other: CLP 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).
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.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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.

 

The relative mean viability of the test item treated tissues was 96.1% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 3.9% relative to the negative control treated tissues and the standard deviation value of the viability was 1.3%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.846 and the standard deviation value of the viability was 6.8%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 13.8%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

 

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

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 30 to February 4, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 439.
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
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on August 21, 2018 / Signed on November 19, 2018)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH top-down 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): 19-EKIN-005
- Production date: not reported
- Shipping date: not reported
- Delivery date: 29 January 2019
- Expiry date: 4 February 2019
- Date of initiation of testing: 30 January 2019

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 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each wellsidual 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:
Deviation 1
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

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 = 1.9 mg/ml ( ≥ 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
absence of HIV1 & 2 & hepatitis C antibodies; absence of hepatitis B antigen HBs
- 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 six months. This was taken to show the correct functioning of the test system.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

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

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 µL

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 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:
81.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
3.7%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: yes, an assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.839 and the standard deviation value of the viability was 1.1%. 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 3.7% relative to the negative control treated tissues and the standard deviation value of the viability was 1.1%. 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 1.8%. The test item acceptance criterion was therefore satisfied.

Table 7.3.1/1: EpiSkin™ results

Item

OD570of tissues

Mean OD570of triplicate tissues

±SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.847

0.839

0.009

101.0

100*

1.1

0.839

100.0

0.830

98.9

Positive Control Item

0.037

0.031

0.010

4.4

3.7

1.1

0.020

2.4

0.036

4.3

Test Item

0.680

0.682

0.015

81.0

81.3

1.8

0.668

79.6

0.698

83.2


OD=Optical Density

SD=       Standard deviation

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

Interpretation of results:
other: CLP 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).
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.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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.

 

The relative mean viability of the test item treated tissues was 81.3% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 3.7% relative to the negative control treated tissues and the standard deviation value of the viability was 1.1%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.839 and the standard deviation value of the viability was 1.1%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.8%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.

 

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

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:
24 April 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted prior to the adoption of the OECD test Guideline No. 437 but followed the SOP used to validate the model. The absence of references to historical control data reduce the reliability of this assay, but this deviation is considered not to have affected the conclusions (unequivocal results)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
historical control data are not reported, some information on eye collection and preparation are missing
Qualifier:
according to guideline
Guideline:
other: INVITTOX (UK) protocol no.98 "The Bovine Corneal Opacity and Permeability Assay"
Version / remarks:
February 1994
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, D-64395 Brensbach
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes and they were contained and transported in Hank’s BSS supplemented with streptomycin / penicillin at room temperature.
- Time interval prior to initiating testing: The bovine eyes were fetched from the slaughterhouse and stored in the refrigerator at about 2 - 8 °C until preparation of the corneas on the same day,
- indication of any existing defects or lesions in ocular tissue samples: none reported. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Hank’s BSS supplemented with streptomycin / penicillin
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):1 mL of test item was applied on each cornea.
Duration of treatment / exposure:
10 minutes ± 30 seconds at 32 ± 2 °C in a horizontal position
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above.
Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the Oring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete minimum essential medium (cMEM). The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 2 °C in a water-bath.
At the end of the incubation period, the medium was removed from both compartments and replaced with fresh cMEM, and the basal opacity was determined (t0). For measurement, the posterior compartment was plugged and the anterior compartment was unplugged.
The bovine eyes were fetched from the slaughterhouse and stored in the refrigerator at about 2 - 8 °C until preparation of the corneas on the same day,
The cornea of the freshly delivered eye was removed as described earlier and inserted in pre-cooled preservation medium. The corneas were stored individually in a minimum volume of 5 mL. The preservation medium was composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.

QUALITY CHECK OF THE ISOLATED CORNEAS : macroscopic examination

NUMBER OF REPLICATES : 3 corneas/group

NEGATIVE CONTROL USED : 0.9% (v/v) saline

POSITIVE CONTROL USED : Ethoxyethanol

APPLICATION DOSE AND EXPOSURE TIME : Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item or negative or positive control at a volume of 1.0 mL on the surface of the corneas and was incubated at 32 ± 2 °C in the water-bath, while the corneas were in a horizontal position.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE:
- Number of washing steps after exposure period: After the test item was rinsed off from the application side by changing cMEM several times, in minimum three times, fresh cMEM was added and opacity was measured (t10).

- POST-EXPOSURE INCUBATION: The corneas were then incubated at 32 ± 2 °C for further two hours in a vertical position, followed by a third opacity reading (t120).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer determines changes in the light transmission passing through the corneas, and displays a numerical opacity value. This value was recorded in a table. The opacitometer was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After recording the basal opacity of all corneas, the mean value of all corneas were taken and any cornea deviating from this by more than 3 units, also –3 units is possible, was discarded. Sets of three corneas were used for treatment with the test item and the negative and positive controls.
- Corneal permeability: Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (v/v) fluorescein solution. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32 ± 2 °C. Medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
0-3 Non eye irritant
3.1-25 Mild eye irritant
25.1-55 Moderate eye irritant
55.1-80 Severe eye irritant
> 80.1 Very severe eye irritant
Irritation parameter:
in vitro irritation score
Run / experiment:
mean/3 corneas
Value:
0.13
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS = 0.92
Positive controls validity:
valid
Remarks:
IVIS = 45.06
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: the test item did not cause any opacity or permeability of the corneas compared with the results of the negative control

DEMONSTRATION OF TECHNICAL PROFICIENCY: not included in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: cannot be assessed
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not reported

Table 7.3.2/1: Individual and Mean Corneal Opacity and Permeability Measurements

Test Group

Opacity value =

Difference (t120-t0) of Opacity

Permeability at

490 nm (OD490)*

In vitro Score

Mean in vitroScore

Proposed in vitro Irritation Scale

 

 

Mean

 

Mean

 

 

 

Negative

Control

0

0

0.049

0.061

0.74

0.92

Non eye irritant

Negative

Control

0

0.046

0.69

Negative

Control

0

0.088

1.32

Positive control

32

0.566

40.49

Moderate eye irritant

Positive control

30

1.180

47.70

Positive control

28

1.265

46.98

Test item

0

0.000

-1.05

Non eye irritant

Test item

0

0.104

1.56

Test item

0

0.059

-0.12

 

Assay validity

The positive control elicited an In Vitro Irritancy Score of 45,06. Even if below the cut-off value of 53, it is close to the treshold and the since the negative control and test item results were not equivocal, the study was considered to be acceptable.

Interpretation of results:
GHS criteria not met
Conclusions:
With an IVIS < 3, the test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 and to the GHS.
Executive summary:

An in vitro eye irritation study was performed prior to the adoption of the OECD Guideline 437 (but follows the SOP used to validate the model), and in compliance with GLP to assess the corneal damage potential of test substance by means of the BCOP assay using fresh bovine corneae. 

After a first opacity measurement of the fresh bovine corneas (t0), the neat test item , the positive, and the negative controls were applied to corneas and incubated for 10 minutes at 32 ± 2 °C in cMEM (complete Minimum Essential Medium), supplemented with 10% FCS (Fetal Calf Serum). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t10). Further, the corneas were incubated for another 120 minutes at 32 ± 2 °C in medium, and opacity was measured a third time (t120).

The opacity measurement permeability of the corneas was determined by application of 1 mL of a fluorescein solution for about 90 minutes at 32 ± 2 °C. The liquid in the posterior chamber was measured spectrophotometrically.

The negative control (0.9% NaCl solution) showed neither an increase of opacity nor permeability of the corneas. The in vitro score was calculated as 0.92.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneas and therefore, is classified as moderate eye irritant. The in vitro score was calculated as 45.06. The positive controlelicited an In Vitro Irritancy Score of 45.06. Even if below the cut-off value of 53, it is close to the treshold and the since the negative control and test item results were not equivocal, the study was considered to be acceptable.

The test item did not cause any opacity or permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 0.13 and therefore, the test item was classified as non eye irritant.

 

With an IVIS < 3, the test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 and to the GHS.

The study is acceptable and satisfies the requirement for an in vitro eye irritation assay.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

A key study was identified for skin corrosion. This in vitro skin corrosion study (RCC, 2008, Rel.2) 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 (Item 2) treated tissues was 92.6% and 85.5% after 3 and 60 minutes exposure, respectively. 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 not to be corrosive to skin.

The testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the skin 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

No local effects were reported in acute oral study at 2000 mg/kg bw (in PEG 300)

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

The substance is not corrosive (OECD 431, GLP, Rel.2)

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 (Top-down strategy) on Items 1, 4 and 5. The substances are not irritant to 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?

NO

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 top-down, in vitro skin irritation studies (Envigo, 2017 / Covance, 2019 / Covance, 2019) were performed on Items 1, 3 and 4 according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN reconstructed human epidermis model.

The relative mean viability of the test item treated tissues was 95.9% for Item 1, 96.1% for Item 4 and 81.3% for Item 5, after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The quality criteria required for acceptance of results in the test were satisfied.With a tissue viability > 50%, the test items were considered to be not irritant to skin.

Eye irritation:

A key study was identified on Item 2 (RCC, 2008, rel.2). This in vitro eye irritation study was performed prior to the adoption of the OECD Guideline 437 (but follows the SOP used to validate the model), and in compliance with GLP to assess the corneal damage potential of test substance by means of the BCOP assay using fresh bovine corneae.  

The negative control (0.9% NaCl solution) showed neither an increase of opacity nor permeability of the corneas. The in vitro score was calculated as 0.92.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneas and therefore, is classified as moderate eye irritant. The in vitro score was calculated as 45.06. The positive controlelicited an In Vitro Irritancy Score of 45.06. Even if below the cut-off value of 53, it is close to the treshold and the since the negative control and test item results were not equivocal, the study was considered to be acceptable.

The test item did not cause any opacity or permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 0.13 and therefore, the test item was classified as non eye irritant. 

With an IVIS < 3, the test item does not require classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data no additional self-classification is proposed for the substance (all qualities, see Section 13 for further details on composition) regarding skin and eye irritation according to the CLP and to the GHS.

No data was available regarding respiratory irritation, however the substance not being classified for skin and eye irritation, no classification is expected for respiratory irritation.