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Administrative data

Description of key information

- In an in vitro skin irritation study (OECD 439) test item was considered as non irritant. (GLP study, Rel.1, K)

- In an in vitro eye irritation study (OECD 437) test item was considered as non irritant. (GLP study, Rel.1, K)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 18 to 23, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February
2009), ECVAM Skin Irritation Validation Study.
Deviations:
not applicable
Qualifier:
equivalent or similar to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Test system:
human skin model
Source species:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed epidermises
Details on animal used as source of test system:
Not relevant
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE:
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 17 February 2015
Expiry date: 23 February 2015
EpiSkinTM Tissues (0.38cm2) lot number: 15-EKIN-007
Maintenance Medium lot number: 15-MAIN3-007
Assay Medium lot number: 15-ESSC-007

MAIN TEST:
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. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to moisten the tissue culture surface to assist test item removal (see discussion). 10 µL (26.3 mg/cm2) of the test item was then applied to the epidermal surface. .

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps. For the test item treated tissues the test item was peeled off the tissue culture surface using a pair of sterile forceps prior to rinsing. The tissues were 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 oC, 5% CO2 in air for 42 hours.

MTT LOADING/FORMAZAN EXTRACTION (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.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The 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.

VIABILITY CALCULATION:
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1. The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- The test item was warmed to 70 °C to soften and then allowed to cool to 37 °C before use.
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): Undiluted

The Test Item was a solid block and extremely sticky when at the using temperature of 37oC.
Test Item left adhered to the tissues would have been expected to have resulted in interference with the Optical Density measurements rendering the results unusable.
It was therefore considered that moistening the tissue culture surface with 5 μL of sterile water prior to application of the Test Item (as the method for application of solids) would increase the chance of adequate removal at the end of the exposure period without compromising the validity of the study.
At the end of the exposure period (rinsing) the Test Item was still required to be peeled off the tissue culture surface but not stuck fast (unable to remove). The fact that the Test Item need to be peeled away was considered to be evidence that adequate contact was achieved and the validity of the study was not compromised.
Duration of treatment / exposure:
15 minutes
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):
42 hours post-exposure period
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
105.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

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.

 

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

0.967

0.929

0.03

104.1

100*

3.5

0.912

98.2

0.909

97.8

Positive Control Item

0.063

0.065

0.02

6.8

7.0

1.7

0.050

5.4

0.082

8.8

Test Item

1.089

0.984

0.12

117.2

105.9

13.2

0.849

91.4

1.013

109.0

 

SD=Standard deviation; *= The mean viability of the negative control tissues is set at 100%; OD562= Optical Density

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 7.0% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.7%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.929 and the standard deviation value of the percentage viability was 3.5%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 13.2%. The test item acceptance criterion was therefore satisfied.

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

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

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 105.9% after the 15-Minute exposure period and 42 hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

Under the test conditions, test material is not classified according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 19, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 437 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Species:
other: Bovine eye
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Test Item Formulation and Experimental Preparation: The test item was warmed and melted to 70 °C and then allowed to cool to 32 °C before use.
- Amount(s) applied (volume or weight with unit): 0.75 mL was applied on each cornea
Duration of treatment / exposure:
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
Duration of post- treatment incubation (in vitro):
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
Details of test procedure:
- Treatment of corneas: Corneas obtained from freshly slaughtered adult cattle (from a local abattoir) were mounted in corneal holders. Both chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and incubated for 60 minutes at 32 ± 1 °C. Before the treatment, opacity measurement was performed using an opacitometer. MEM was removed from anterior chamber and the test item (0.75 mL) was applied on each cornea. The holders were incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.
- Application of sodium fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the posterior and anterior chambers were removed, the posterior chamber is replaced with fresh complete MEM and the anterior chamber is replaced with 1 mL of sodium fluorescein solution 4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.
- Permeability determinations: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- In Vitro Irritancy Score (IVIS) for test item, negative and positive controls were 0.2, 1.9 and 46.2, respectively.
- Corneal Epithelium Condition: The corneas treated with the test item were clear 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

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 27.8 – 51.0. The positive control acceptance criterion was therefore satisfied.

 

The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test item is not classified as eye irritant according to the Regulation (EC) No. 1272/2008 and to the GHS.
Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 120 minutes. Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). Before the treatment, a first opacity measurement was performed using an opacitometer.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 test item, negative and positive control induced an IVIS of 0.2, 1.9 and 46.2, respectively. The corneas treated with the test item were clear 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.

 

The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0, therefore the acceptance criterion was satisfied. The negative control gave opacity of ≤4.7 and permeability ≤0.080, therefore the acceptance criterion was satisfied.

 

Under the test conditions, test item is not classified as eye irritant according to the Regulation (EC) No. 1272/2008 and the Directive 67/548/EEC and to the GHS.

This study is considered as acceptable and satisfies 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:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTMreconstructed 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 105.9% after the 15-Minute exposure period and 42 hours post-exposure incubation period.The quality criteria required for acceptance of results in the test were satisfied.

 

Eye irritation:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 120 minutes. Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). Before the treatment, a first opacity measurement was performed using an opacitometer.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 test item, negative and positive control induced an IVIS of 0.2, 1.9 and 46.2, respectively. The corneas treated with the test item were clear 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.

 The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0, therefore the acceptance criterion was satisfied. The negative control gave opacity of ≤4.7 and permeability ≤0.080, therefore the acceptance criterion was satisfied.

 

Justification for classification or non-classification

Harmonized classification:

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

 

Self classification:

Skin irritation:

Based on the available data:

- no additional self-classification is proposed regarding skin irritation according to the CLP and to the GHS.

 

Eye irritation:

Based on the available data, no additional self-classification is proposed regarding eye irritation according to the CLP and to the GHS.

 

Respiratory irritation:

No data was available regarding respiratory irritation.