Registration Dossier

Administrative data

Description of key information

Skin irritant Category 2 (OECD 439, K, Rel.1)

Eye irritant Category 2A (OECD 405, 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:
04 December 2014 to 09 April 2015
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 deviations: historical data and tissues certificate are not reported
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
historical data and tissues certificate are not reported
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
historical data and tissues certificate are not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on 12-14 March 2014 / Signed on 12 May 2014)
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: no data
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 03 December 2014
EpiSkinTM Tissues (0.38cm2) lot number: 14-EKIN-046
Maintenance Medium lot number: 14-MAIN3-052
Assay Medium lot number: 14-ESSC-049

TEST FOR DIRECT MTT REDUCTION
- 10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
- If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

PRE-INCUBATION (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

APPLICATION OF TEST ITEM AND RINSING (DAY 1)
- 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 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 well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE (DAY 3)
- Following the 42 - hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
- MTT concentration: Tissues were transferred to the MTT filled wells (0.3 mg/mL MTT solution)
- Incubation time: Tissues were incubated for 3 hours at 37 °C, 5% CO2 in air
- At the end of the 3 - hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6)
- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

PREDICTION MODEL / DECISION CRITERIA
Classification of irritation potential is based upon relative mean tissue viability following the 15 - minute exposure period followed by the 42 - hour post-exposure incubation period
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-Irritant
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL (26.3 μL /cm2)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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of SDS 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 hours.
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: 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:
9.9
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:
TEST ITEM
The relative mean viability of the test item treated tissues was 9.9% after a 15 - minute exposure period and 42 hours post-exposure incubation period.
The mean concentration of inflammatory mediator IL-1α in the culture medium retained from the test item treated tissues was 164.375 pg/mL.

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.868 and the standard deviation value of the percentage viability was 1.3%. 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 10.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.4%. 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 test item treated tissues was 1.8%. 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

0.871

0.868

 0.011

100.3

100*

1.3

0.878

101.2

0.856

98.6

Positive Control Item

0.082

0.093

0.012 

9.4

10.7

1.4

0.106

12.2

0.090

10.4

Test Item

0.080

0.086

 0.015

9.2

9.9

1.8

0.074

8.5

0.103

11.9

 

SD = Standard deviation

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

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 skin irritant (Category 2) according to Regulation (EC) No 1272/2008 (CLP) and to GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN 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 9.9% after the 15-Minute exposure period and 42 hours post-exposure incubation period.

The mean concentration of inflammatory mediator IL-1α in the culture medium retained from the test item treated tissues was 164.375 pg/mL.

 

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

 

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-14 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 431 with minor deviations: Historical data and tissues certificate are not reported.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2014
Deviations:
yes
Remarks:
Historical data and tissues certificate are not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 17 June 2015 / signed on 24 September 2015)
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen
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
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Date received: 11 August 2015
- EpiDermTM Tissues (0.63cm2) lot number: 21685
- Assay Medium lot number: 080615ZSD
Upon receipt of the Epiderm™ tissues, the sealed 24-well plate was stored in a refrigerator until use.

TEST FOR DIRECT MTT REDUCTION
- 50 μL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.

ASSESSMENT OF COLOR INTERFERENCE WITH THE MTT ENDPOINT
- 50 μL of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

PRE-INCUBATION
- The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

APPLICATION OF TEST ITEM AND RINSING
- Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre-incubation of the EpiDerm™ tissues, duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. 50 µL of the test item was applied topically to the corresponding tissues.

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of exposure period, each tissue was rinsed using DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: Tissues were incubated for 3 hours at 37 °C, 5% CO2 in air
- After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS
- After extraction, 3 x 200 μL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

PREDICTION MODEL / DECISION CRITERIA
Classification of corrosivity potential is based on relative viabilities for both exposure times
- 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:
99.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
45.6
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:
TISSUE VIABILITY:
The relative mean viability of the test item treated tissues was 99.4 and 45.6% 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 4.4 and 3.9% after 3 and 60 minutes exposure, respectively.

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.
- 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.429 for the 3 minute exposure period and 2.369 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 3.9% 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 OD562 of individual

tissues

Mean OD562 of duplicate

tissues

Standard Deviation

Coefficient of Variation (%)

Relative Mean Viability (%)

Negative

Control

3 Minutes

2.504

2.429

0.107

4.4

100*

2.353

60 Minutes

2.358

2.369

0.016

0.7

2.380

Positive

Control

3 Minutes

0.128

0.107

0.030

NA

4.4

0.085

60 Minutes

0.106

0.093

0.018

NA

3.9

0.080

Test Item

3 Minutes

2.529

2.414

0.163

6.8

99.4

2.298

60 Minutes

1.221

1.080

0.200

18.5

45.6

0.938

 

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:
other: Non-corrosive
Conclusions:
The test item was considered to be non-corrosive to the skin, according to EU CLP Regulation (EC) No 1272/2008 and GHS.
Executive summary:

An in vitro skin irritation 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. 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 MTT solution containing the test item did not turn blue and indicate the test item did not reduce MTT. The solution containing the test item did not become colored and indicate the test item did not have the potential to cause color interference.

 

The relative mean viability of the test item treated tissues was 99.4 and 45.6% 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 4.4 and 3.9% 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 GHS.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October to 24 November 2015
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.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 17 June 2015 / signed on 24 September 2015)
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- Storage condition of test material: Refrigerated at 4°C in the dark under nitrogen
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Leicestershire, UK.
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 3.05-3.50 kg
- Housing: Animals were individually housed in suspended cages.
- Diet: Food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum.
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 26 October to 24 November 2015
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, 72 h and 7, 14 days after instillation of test item
Number of animals or in vitro replicates:
3 males
Details on study design:
PRETREATMENT
- 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. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made.
- 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 hours later. No further analgesia was required.
- After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 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. In order to confirm the absence or presence of corneal opacity, examination under ultra violet light following treatment with Sodium Fluorescein BP was performed in one treated eye at the 24 and 48 hour observations. The cornea, conjunctivae and iris were also examined for lesions. The control eyes were also similarly treated with Sodium Fluorescein BP.

OTHERS
- Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Ocular reactions:
- Scattered or diffuse corneal opacity was noted in all treated eyes at the 24, 48 and 72 hour observations.
- Iridial inflammation was noted in all treated eyes 1 hour after treatment and at the 24, 48 and 72 hour observations.
- Moderate conjunctival irritation was noted at all treated eyes 1 hour after treatment and at the 24 and 48 hour observations. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 72 hour observation. Minimal conjunctival irritation was noted in all treated eyes at the 7-Day observation.
- All treated eyes appeared normal at the 14-Day observation.
Other effects:
Body weight: All 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

1 / 1 / 1

2 / 2 / 2

2 / 2 / 2

2 / 2 / 2

24 h

1 / 1 / 1

1 / 2 / 1

1 / 1 / 1

2 / 2 / 2

2 / 2 / 2

2 / 2 / 2

48 h

1 / 1 / 1

2 / 2 / 1

1 / 1 / 1

2 / 2 / 2

2 / 2 / 1

1 / 1 / 1

72 h

1 / 1 / 1

2 / 2 / 2

1 / 1 / 1

2 / 2 / 2

1 / 1 / 1

1 / 1 / 0

D7

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

1 / 1 / 1

1 / 1 / 1

0 / 1 / 0

D14

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

0 / 0 / 0

Mean 24, 48 and 72 h

1.0 / 1.0 / 1.0

1.7 / 2.0 / 1.3

1.0 / 1.0 / 1.0

2.0 / 2.0 / 2.0

1.7 / 1.7 / 1.3

1.3 / 1.3 / 1.0

Reversibility

Completely

reversible

Completely

reversible

Completely

reversible

Completely

reversible

Completely reversible

Completely reversible

Average time (unit) for reversion

7 days

7 days

7 days

14 days

14 days

14 days

Interpretation of results:
Category 2A (irritating to eyes) based on GHS criteria
Conclusions:
Under the test conditions, the test item was classified as Category 2 and 2A (irritating to eyes) according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), respectively.
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 male New Zealand White(Hsdlf:NZW) strain rabbits. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test item, 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, 14 days after instillation of test item and graded according to the Draize method.

 

Scattered or diffuse corneal opacity was noted in all treated eyes at the 24, 48 and 72 hour observations. Iridial inflammation was noted in all treated eyes 1 hour after treatment and at the 24, 48 and 72 hour observations. Moderate conjunctival irritation was noted at all treated eyes 1 hour after treatment and at the 24 and 48 hour observations. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 72 hour observation. Minimal conjunctival irritation was noted in all treated eyes at the 7-Day observation. All treated eyes appeared normal at the 14-Day observation.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 1.0 / 1.0 / 1.0 for cornea score;1.0 / 1.0 / 1.0 for iris score; 2.0 / 2.0 / 2.0 for conjunctivae score and 1.7 / 1.7 / 1.3 for chemosis score.

 

Under the test conditions, the test item was classified as Category 2 and 2A (irritating to eyes) according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

A key study was identified. In this in vitro skin irritation study performed according to the OECD guideline 439 and in compliance with GLP, using the EPISKIN™ 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.

The relative mean viability of the test item treated tissues was 9.9% after the 15-Minute exposure period and 42 hours post-exposure incubation period, which is < 50% therefore the substance is considered as a skin irritant.

The mean concentration of inflammatory mediator IL-1α in the culture medium retained from the test item treated tissues was 164.375 pg/mL.

Due to the positive result obtained in the in vitro skin irritation study, the corrosion potential of the substance was evaluated. In the in vitro skin corrosion study 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. 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 MTT solution containing the test item did not turn blue and indicate the test item did not reduce MTT. The solution containing the test item did not become colored and indicate the test item did not have the potential to cause color interference. 

The relative mean viability of the test item treated tissues was 99.4 and 45.6% 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 4.4 and 3.9% 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 GHS.

Conclusion: skin irritant (Cat.2)

Eye irritation

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, the undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The corneas treated with the test item were slightly cloudy post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In Vitro Irritancy Score (IVIS) for test item, negative and positive controls were 13.7, 1.9 and 49.6, respectively.

The negative and positive controls acceptance criteria were satisfied.

Based on the In Vitro Irritancy Score of 13.7, no prediction of eye irritation can be made (UN GHS/EU CLP).

Therefore, an in vivo study was performed (Envigo, 2016b, rel.1). In this in vivo eye irritation study performed according to the OECD 405 Guideline and in compliance with GLP, 0.1 mL of undiluted substance was instilled into the right eye of 3 male New Zealand White strain rabbits. The upper and lower eyelids were held together for about one second immediately after application, to prevent loss of the test item, and then released. The left eye remained untreated and served as control.

Scattered or diffuse corneal opacity was noted in all treated eyes at the 24, 48 and 72 hour observations. Iridial inflammation was noted in all treated eyes 1 hour after treatment and at the 24, 48 and 72 hour observations. Moderate conjunctival irritation was noted at all treated eyes 1 hour after treatment and at the 24 and 48 hour observations. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 72 hour observation. Minimal conjunctival irritation was noted in all treated eyes at the 7-Day observation. All treated eyes appeared normal at the 14-Day observation.

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 1.0 / 1.0 / 1.0 for cornea score;1.0 / 1.0 / 1.0 for iris score; 2.0 / 2.0 / 2.0 for conjunctivae score and 1.7 / 1.7 / 1.3 for chemosis score.

Under the test conditions, the test substance is classified as irritating to eyes.

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

Based on the available data, additional self-classification is proposed regarding both skin and eye irritation:

- classified as skin irritant Category 2 according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS

- classified as eye irritant Category 2 according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and Category 2A according to the GHS

No data was available regarding respiratory irritation.