Registration Dossier

Administrative data

Description of key information

Skin irritation: irritating (OECD 439, GLP, K, Rel.1)

Skin corrosion: not corrosive (OECD 431, GLP, K, Rel.1)

Eye irritation: not irritating (ICE, GLP, K, Rel.1 & OECD 405, GLP, K, Rel.1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-27 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 439 with minor deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
historical data are not reported
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
historical data are not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)
Specific details on test material used for the study:
- Purity test date: 30 October 2013
- Storage Conditions: ca. 4 °C in the dark under nitrogen
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
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): 14-EKIN-001
- Production date: not reported
- Shipping date: 21 January 2014
- Delivery date: 21 January 2014
- Expiry date: 27 January 2014
- Date of initiation of testing: 22 January 2014

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: not reported
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in DPBS
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: not applicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 1.030, 0.970 and 0.939 (historical control not reported).
- Barrier function: IC50= 2.1 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: water-killed tissues
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared by placing untreated EPISKIN™ 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. In addition to the normal test procedure, the MTT reducing test item was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated. The untreated water-killed controls showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.
- N. of replicates : 3
- Method of calculation used: Relative mean viability (%) = (Mean OD562 of test item / Mean OD562 of negative control) x 100

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is greater than 50%
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 of the test item was applied to the epidermis surface.
- Concentration (if solution): Undiluted

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of SDS
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes at room temperature.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.
Duration of post-treatment incubation (if applicable):
- On Day 3, at the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- On Day 6, at the end of the formazan extraction period: 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 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Number of replicates:
Triplicate tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period
Value:
8.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- Direct MTT Reduction: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results showed a negligible degree of interference due to direct reduction of MTT occurred (OD562 of the treated killed tissues = 0.005 following subtraction of the untreated killed tissues; Treated killed tissues = 0.067 – untreated killed tissues = 0.062 = 0.005). It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

- Test item: The relative mean viability of the test item treated tissues was 8.1% after a 15-Minute exposure period and 42 hours post-exposure incubation period. It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.980 and the standard deviation value of the percentage viability was 4.7%. 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 5.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.4%. 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 test item tissues was 1.6%. The test item acceptance criterion was therefore satisfied.

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

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

1.030

0.980

0.046

105.1

100*

4.7

0.970

99.0

0.939

95.8

Positive Control Item

0.050

0.054

0.004

5.1

5.5

0.4

0.058

5.9

0.053

5.4

Test Item

0.064

0.080

0.016

6.5

8.1

1.6

0.095

9.7

0.080

8.2

 

SD = Standard deviation

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

= Control group shared with Harlan study number 41304108

OD562 = Optical Density

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test item is classified as "Category 2 irritant" according to the Annex VI of the 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. 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 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 8.1% after the 15-Minute exposure period and 42 h post-exposure incubation period.

 

The mean OD562 for the negative control treated tissues was 0.980 and the standard deviation value of the percentage viability was 4.7%. The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.4%. The negative and positive controls acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 1.6%. The test item acceptance criterion was therefore satisfied. 

 

Under the experimental conditions of this study, the test item is classified as "Category 2 irritant" according to Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-17 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 431.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
B.40bis of 30 May 2008
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: No data
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE ; Viable Tissues
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Date received: not reported (August, 2016)
- EpiDermTM Tissues (0.63cm2) lot number: 23393
- Assay Medium lot number: not reported
Upon receipt of the Epiderm™ tissues, the sealed 24-well plate was stored in a refrigerator until use.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE : Non-viable Tissues
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Date received: 14 February 2017
- EpiDermTM Tissues (0.63cm2) lot number: 23348
- Assay Medium lot number: 020917TME
Upon receipt of the Epiderm™ tissues, the sealed 24-well plate was stored in a refrigerator until use.

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

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: without reference filter
- Filter bandwidth: filter band pass ± 15
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570 nm) = 1.52 ± 0.144 and 1.850 ± 0.053 for viable and non-viable tissues, respectively (acceptance criteria: between 1.0-3.0)
- Barrier function: ET-50 = 5.55 hours and 6.18 hours for viable and no-viable tissues, respectively (acceptance criteria: between 4.77-8.72 hours)
- Morphology: Tissue viability and the barrier function test are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile (Long term antibiotic and antimycotic free culture)
- Reproducibility: In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.

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

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

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): Undiluted

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:
Tissues were treated with the test item for exposure periods of 3 and 60 minutes.
Duration of post-treatment incubation (if applicable):
- MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- At the end of the formazan extraction period: The optical density was measured at 562 nm using the Anthos 2001 microplate reader.
Number of replicates:
Duplicate tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
113.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
3.5%
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
89.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
2.9%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT. Direct reduction was <30% relative to the negative control and therefore acceptable.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.665 for the 3 minute exposure period and 1.674 for the 60 minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 2.9% relative to the negative control following the 60 minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20-100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

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

  

Tissue Exposure Period Mean OD562of individual tissues (tvt) Mean OD562of duplicate tissues Corrected OD562(tvt - (tkt - ukt)) Standard Deviation Coefficient of Variation (%) Relative Mean Viability (%)
Negative Control 3 Minutes 1.644 1.665   0.029 1.7 100*
1.685  
60 Minutes 1.64 1.674   0.047 2.8
1.707  
Positive Control 3 Minutes 0.073 0.059   0.02 N/A 3.5
0.045  
60 Minutes 0.055 0.049   0.009 N/A 2.9
0.042  
Test item 3 Minutes 1.898 1.967 1.891 0.097 5.1 113.5
2.035
60 Minutes 1.884 1.789 1.501 0.135 9.0 89.7
1.693

3 minute exposure corrected mean OD562 = 0.206 (tkt) - 0.130 (ukt) = 0.076

60 minute exposure corrected mean OD562 = 0.418 (tkt) - 0.130 (ukt) = 0.288

Tvt = Treated viable tissue

Tkt = Treated killed tissue

Ukt = Untreated killed tissue

Relative mean viability (%) = (Mean OD562 of test item / Mean OD562 of negative control) x 100

Coefficient of Variation = (standard deviation / mean OD of duplicate tissues) x 100

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

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test item was considered to be non-corrosive to the skin according to EU CLP Regulation (EC) No 1272/2008 and to the GHS.
Executive summary:

An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EPIDERMTM reconstructed human epidermis model.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. 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 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 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 113.5 and 89.7% after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 3.5 and 2.9% after 3 and 60 minutes exposure, respectively.

 

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

 

The test item was considered to be non-corrosive to the skin, 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 conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March to 14 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 405 without deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)
Specific details on test material used for the study:
- Purity test date: 30 October 2013
- Storage Conditions: ca. 4 °C in the dark under nitrogen
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK.
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.24-2.68 kg
- Housing: Animals were individually housed in suspended cages.
- Diet: Food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum.
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 31 March to 14 April 2014
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
No washing was done
Observation period (in vivo):
1, 24, 48 and 72 h and 7 days after instillation of test material
Number of animals or in vitro replicates:
3 (1 female and 2 males)
Details on study design:
PRE-TREATMENT:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

PROCEDURE:
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre-dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye. A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released.
Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 h later. No further analgesia was required. After consideration of the ocular responses produced in the first treated animal, two additional animals were similarly treated.

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM: Draize scale as described in the OECD guideline No. 405.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24 h
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- No initial pain reaction was noted in any animal following instillation of the test item.
- No corneal effects were noted during the study.
- Iridial inflammation was noted in one treated eye one and 24 h after treatment.
- Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 24 h observation. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in one other treated eye at the 48 h observation. Minimal conjunctival irritation was noted in both of these treated eyes at the 72 h observation.
- One treated eye appeared normal at the 48 h observation and two treated eyes appeared normal at the 7-Day observation.
Other effects:
One animal showed a slight body weight loss, although this was considered to be within the normal variation for this species/strain, and two animals showed expected gain in body weight during the study.

Table 7.3.2/1: Eye irritation response data for each animal at each observation time

Score at time point

Cornea

Iris

(/2)

Conjunctivae

Opacity

(/4)

Area

(/4)

Redness

(/3)

Chemosis

(/4)

Discharge

(/3)

1 h

0 / 0 / 0

0 / 0 / 0

0 / 0 / 1

2 / 2 / 2

1 / 1 / 2

2 / 2 / 2

24 h

0 / 0 / 0

0 / 0 / 0

0 / 0 / 1

1 / 2 / 2

0 / 1 / 1

0 / 1 / 1

48 h

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

0 / 1 / 2

0 / 0 / 1

0 / 0 / 1

72 h

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

0 / 1 / 1

0 / 0 / 1

0 / 0 / 0

D 7

- / 0 / 0

- / 0 / 0

- / 0 / 0

- / 0 / 0

- / 0 / 0

- / 0 / 0

Average 24, 48 and 72 h

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0.3

0.3 / 1.3 / 1.7

0 / 0.3 / 1.0

0 / 0.3 / 0.7

Reversibility

 -

 -

Completely

reversible

Completely

reversible

Completely reversible

Completely reversible

Average time (unit) for reversion

-

-

48 h

7 days

7 days

72 h

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test substance is not classified as irritating to eyes according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In an eye irritation study performed according to the OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test item was instilled into the right eye of 3 New Zealand White rabbits (1 female and 2 males). The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test material, and then released. The left eye remained untreated and served as control. The eyes were examined and the changes were observed at 1, 24, 48, 72 h and 7 days after treatment and graded according to the Draize method.

No initial pain reaction was noted in any animal following instillation of the test item. No corneal effects were noted during the study. Iridial inflammation was noted in one treated eye one and 24 h after treatment. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 24 h observation. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in one other treated eye at the 48 h observation. Minimal conjunctival irritation was noted in both of these treated eyes at the 72 h observation. One treated eye appeared normal at the 48 h observation and two treated eyes appeared normal at the 7-Day observation.

One animal showed a slight body weight loss, although this was considered to be within the normal variation for this species/strain, and two animals showed expected gain in body weight during the study.

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

Under the test conditions, the test substance is not classified as irritating to eyes according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 438 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on 03-04 June 2013 / signed on 13 September 2013
Specific details on test material used for the study:
- Purity test date: 30 October 2013
- Storage Conditions: 6 ± 3 °C
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they were killed for human consumption were used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e.) approximately 7 weeks old, 1.5 - 2.5 kg).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 05 March 2014 at 8:15 a.m. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 05 March 2014 at 10:30 am.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL of the test item was applied, as supplied, to the cornea
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye
Number of animals or in vitro replicates:
Test item: 3 eyes
Positive control: 3 eyes
Negative control: 1 eye
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
- The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the isotonic saline drip. The chambers of the superfusion apparatus was temperature controlled between 31.2 °C and 32.0 °C.
- After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of> 0.5; (ii) corneal opacity> 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
- Once all eyes had been examined and approved, the eyes were incubated between 45 minutes and 63 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 1, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 µL of the test item was applied, as supplied, to the cornea, such that the entire surface of the cornea is evenly covered with the test item. The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. 1. For the measurement of corneal thickness, the slit-width was set, equalling 0.095 mm.
- The endpoints evaluated are corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is determined only at pretreatment and 30 minutes after test item exposure) are determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling is determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
- Mean maximum opacity score: Corneal opacity is calculated by using the area of the cornea that is most densely opacified for scoring. The mean corneal opacity value for all test eyes is calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score is then given for each test or control item
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.
Irritation parameter:
cornea opacity score
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
2.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.0, corresponding to the ICE class I;
- mean score of fluorescein retention: 2.3, corresponding to the ICE class III;
- maximal mean corneal swelling: +3% corresponding to the ICE class I.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 2 x IV, 1 x III. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment

 

Endpoint measured

Eye No.

Time (minutes)

0

30

75

120

180

240

Corneal opacity

9

0

0

0

0

0

0

10

0

0

0

0

0

0

11

0

0

0

0

0

0

Mean

0.0

0.0

0.0

0.0

0.0

0.0

ICE class

I

Fluorescein retention

9

0

2

-

-

-

-

10

0

2

-

-

-

-

11

0

3

-

-

-

-

Mean

0.0

2.3

-

-

-

-

ICE class

III

Corneal thickness

9

0.51

0.51

0.51

0.51

0.51

0.52

10

0.51

0.52

0.52

0.51

0.52

0.52

11

0.53

0.56

0.54

0.55

0.54

0.55

Corneal swelling (%)

9

-

0

0

0

0

2

10

-

2

2

0

2

2

11

-

6

2

4

2

4

Mean

-

3

1

1

1

3

ICE class

I

Combination of the 3 Endpoints

2 x I, 1 x III

Classification

Non "Corrosive / severe irritant"

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the test conditions and at the time of this study, the authors of the study concluded that the test item is not classified as irritating to eyes according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS. However, based on the results of the test and the criteria for classification, the results are inconclusive and therefore no prediction can be made on the basis of this study.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item ST 14 C 12 was applied, as supplied, at the dose of 30 µL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

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

- maximal mean score of corneal opacity: 0.0, corresponding to the ICE class I;

- mean score of fluorescein retention: 2.3, corresponding to the ICE class III;

- maximal mean corneal swelling: +3% corresponding to the ICE class I.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 3 x I. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 2 x IV, 1 x III. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

 

Under the test conditions and at the time of this study, the authors of the study concluded that the test item is not classified as irritating to eyes according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS. However, based on the results of the test and the criteria for classification, the results are inconclusive and therefore no prediction can be made on the basis of this study.

This study is not considered as sufficient to satisfy the requirement for eye irritation endpoint.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

One key study (EpiSkin) was identified but was not sufficient to conclude on the corrosive potential of the registered substance. Since no key study was identified regarding corrosivity 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:

Table 7.3/1: ITS for skin irritation/corrosion

 

Element

Information

Conclusion

Comments

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance is a corrosive
or irritant?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

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

Existing data from general toxicity studies via the dermal route and from sensitisation studies

4a

Is the substance classified as acutely toxic by the dermal route (Category 1)?

NO

 

4b

Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?

NO

In an acute dermal study, Very slight erythema, glossy skin and/or crust formation were noted at the test sites of two out of 10 animals but no sign of irritation was observed in the other eight animals.

4c

Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated
exposure?

NO

No sign of irritation was observed in anin vivoskin sensitisation study (OECD 406, GPMT).

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.

YES

Anin vitroskin irritation study (OECD 439, Episkin™) gave a positive result.Relative mean viability = 8.1% => skin irritant or corrosive

6c

Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?

NO

 

Weight-of- Evidence analysis

7

The “elements” described above may be arranged as appropriate. Taking all available existing and
relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?

NO

 

New in vitro test for corrosivity

8

Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

 

New in vitro test for irritation

9

Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

YES

Following the bottom-up approach, an OECD TG 431 (EpiDerm ™) study was performed. Relative mean viability = 113.5% and 89.7% after 3 and 60 minutes exposure, respectively <=> not corrosive to skin

New in vivo test for corrosion/irritation

10

To be used only as a last resort

NO

 

 

In vitro OECD 439 (Test No. 1)

In the first key study (Harlan, 2014, rel.1), the purpose of this in vitro test was to evaluate the skin irritation potential of the test substance using the EPISKINTM 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 OECD Guideline No. 439 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 8.1%, after the 15 -minute exposure period and 42 hours post-exposure incubation period. With a tissue viability < 50%, the test substance was considered to be irritant to skin.

In vitro OECD 431 (Test No. 2)

In the second key study (Envigo, 2017, rel.1), the purpose of this in vitro test was to evaluate the skin corrosion potential of the test substance using the EPIDERMTM reconstructed human epidermis model. This in vitro study was performed according to the OECD Guideline 431 and in compliance with GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes.

Negative and positive control groups were treated for each exposure period.

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 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 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 113.5 and 89.7% after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 3.5 and 2.9% after 3 and 60 minutes exposure, respectively.

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

The test item was considered to be non-corrosive.

According to the available data for skin irritation/corrosion, the test substance is considered to be irritant for the skin.

 

Eye irritation:

Two key studies were identified (Test No. 1 and 2).

Table 7.3/2: Summary of eye irritation tests

 

Test n°

Test / Guideline

Reliability

Focus

Results

1

 

Phycher, 2014

Isolated Chicken Eye test method

(OECD 438)

K, rel. 1

Eye irritation

no prediction can be made

2

 

Harlan, 2014

Acute eye irritation/corrosion (OECD 405)

K, rel. 1

Eye irritation

 non irritant

- in vitro OECD 438 (Test No. 1):

An in vitro study was available on the substance itself regarding eye irritation. In this in vitro eye corrosion study performed according to the OECD Guideline 438 and in compliance with GLP, the test item was applied, as supplied, at the dose of 30 μL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test substance were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were: Maximal score of corneal opacity: 0.0, corresponding to ICE class I; Mean score of fluorescein retention: 2.3, corresponding to ICE class III; Maximal corneal swelling: +3% at 30 minutes post dose corresponding to ICE class I.

As expected the positive and negative control were classified as “Corrosive/Severe Irritant” and "No category" respectively.

However, considering the results obtained, it would require additional testing (in vitro and/or in vivo) to establish a classification as “irritant” (category 2) or as “no category”.

- in vivo OECD 405 (Test No. 2):

An in vivo test was therefore performed due to worldwide registration needs. In this eye irritation study performed according to the OECD guideline 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into one eye of 3 female New Zealand White rabbits. The other eye remained untreated and served as control. The upper and lower eyelids were held together for about one second immediately after instillation. The eyes were examined and the changes were observed at 1, 24, 48 and 72 h after treatment and graded according to the Draize method.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.0/0.0/0.0 for the corneal opacity and iridial inflammation, 1.7/0.7/0.7 for conjunctival redness and 1.0/0.3/0.3 for chemosis. The effects observed were all reversible within 7 days. The substance is therefore not classified for eye irritation.

According to the available data for eye irritation, the test substance is considered to be non irritant for the eye.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data, the substance is classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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

No data was available regarding respiratory irritation.