Registration Dossier

Administrative data

Description of key information

Assessment of irritancy effects to skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 13 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with current OECD guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Species:
other: Reconstructed Human Epidermis (RHE) Model
Strain:
not specified
Details on test animals and environmental conditions:
The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKINTM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.Skin corrosion refers to the production of irreversible tissue damage in the skin following theapplication of a test item (as defined by the Globally Harmonised System (GHS) for the Classification and Labelling of Chemical Substances and Mixtures). The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safehandling, packing and transport of chemicals. Validation studies have shown that testsemploying human skin models are able to reliably distinguish between known skin corrosivesand non-corrosives (Botham et al., 1995, Barrett et al., 1998 and Fentem et al., 1998). At its 10th Meeting, held on 31 March 1998 at ECVAM, Ispra, Italy, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the EPISKINTM model as scientifically validated for use as a replacement for the animal test. The EPISKINTM model is able to distinguish betweencorrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.The procedure followed is based on the recommended EpiSkinTM Skin Corrosivity Test protocol INVITTOX No 118. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKINTM model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.
Type of coverage:
other: in vitro model
Preparation of test site:
other: in vitro model
Vehicle:
unchanged (no vehicle)
Remarks:
in vitro model
Controls:
not required
Amount / concentration applied:
50 µL of the test item was added to 2.0 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium.
Duration of treatment / exposure:
3, 60 and 240 minutes
Observation period:
3 days (see below)
Number of animals:
none - in vitro method
Details on study design:
Reference Items Information as provided by the suppliers.Identification:0.9% w/v sodium chloride solutionBatch:300999 104Purity:0.9%Expiry Date:01 January 2015Storage Conditions:room temperatureIdentification:Glacial acetic acidBatch:114957Purity:>99.7%Expiry Date:08 November 2014Storage Conditions:room temperatureMATERIALS AND METHODSTest SystemEPISKIN™ Reconstructed Human Epidermis Model KitSupplier : SkinEthic Laboratories, Lyon, FranceDate received : 10 September 2013EpiSkinTM Tissues (0.38cm2) lot number : 13-EKIN-031Maintenance Medium lot number : 13-MAIN3-038Assay Medium lot number : 13-ESSC-032Test Item Formulation and Experimental PreparationThe test item was used as supplied.Preparation of Negative and Positive Control ItemsThe negative control item was used as supplied. The positive control item was used as supplied.Pre-Test ProcedureAssessment of Direct Test Item Reduction of MTTMTT Dye Metabolism, Cell Viability AssayThe MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.Test for Direct MTT ReductionAs specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:50 µL of the test item was added to 2.0 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 corrosion 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 : Yes2.0 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into two wells of the first column of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37 deg C, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.Main TestApplication of Test Item and Rinsing (Day 2)2.0 mL of assay medium, warmed to approximately 37 deg C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 50 µL of the test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 µL of 0.9% w/v Quality CriteriaThe results of the assay are considered acceptable if the following assay acceptance criteria are achieved:Negative Control:The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥0.600 and ≤ 1.500.Positive Control:The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240-Minute exposure period.sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ±5 minutes at 37 deg 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 taken using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.Absorbance/Optical Density Measurements (Day 3)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 L samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 L of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at562 nm (without a reference filter) using the Anthos 2001 microplate reader.Interpretation of ResultsQuantitative MTT Assessment (percentage tissue viability)The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:Relative mean visibility (%): (mean OD562 of test item / mean OD562 of negative control) x 100
Irritation / corrosion parameter:
other: Mean OD562 values
Remarks:
relative mean viabilities
Run / experiment:
3 minutes time point
Value:
114.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean OD562 values
Remarks:
relative mean viabilities
Run / experiment:
60 minutes time point
Value:
107.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean OD562 values
Remarks:
relative mean viabilities
Run / experiment:
240 minutes time point
Value:
120.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Direct MTT ReductionThe MTT solution containing the test item remained yellow. This was taken to indicate that the test item did not reduce MTT.Test Item, Positive Control Item and Negative Control ItemMean OD562 values and viabilities for the negative control, positive control and test item are given in Table 1 below.
Quality CriteriaThe relative mean tissue viability for the positive control treated tissues was 3.6% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 0.868. The negative control acceptance criterion was therefore satisfied.

The relative mean viabilities of the test item treated tissues were as follows:

240 minutes exposure

:

120.4%

60 minutes exposure

:

107.5%

3 minutes exposure

:

114.9%

Table1     Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

Exposure Period

Mean OD562of individual tissues

Mean OD562of duplicate tissues

Relative mean viability (%)

Negative Control Item

240 Minutes

0.871

0.868

100*

0.865

Positive Control Item

240 Minutes

0.029

0.031

3.6

0.032

Test Item

240 Minutes

1.118

1.045

120.4

0.972

60 Minutes

0.925

0.933

107.5

0.941

3 Minutes

0.989

0.997

114.9

1.004

 

Å


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

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-corrosive to the skin. The following classification criteria apply:EU DSD (67/548/EEC): Not classified for corrosivity.EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity. UN Packing Group: Non-Corrosive.
Executive summary:

The test item was classified as non-corrosive to the skin. The following classification criteria apply:

 EU DSD (67/548/EEC): Not classified for corrosivity.

 EU CLP (1272/2008/EC)/ UNGHS: Not classified for corrosivity.

UN Packing Group:Non-Corrosive.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-14 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with current OECD guidelines
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Species:
other: EPISKIN™ Reconstructed Human Epidermis
Strain:
not specified
Details on test animals and environmental conditions:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentem et al., 2001, Zuang et al.,2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.
Type of coverage:
other: in vitro model
Preparation of test site:
other: in vitro model
Vehicle:
other: in vitro model
Controls:
no
Amount / concentration applied:
200 µ L
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes
Observation period:
42 hours
Number of animals:
none - in vitro method
Details on study design:
Reference Items
Negative Control
Information as provided by the supplier
.Identification:Phosphate Buffered Saline Dulbecco’s (PBS)with Ca++ and Mg++
Batch:1188811
Purity:unknown
Expiry Date:01 July 2014
Storage Conditions:approximately 4oC in the dark
Positive Contro
lInformation as provided by the supplier.
Identification:Sodium Dodecyl Sulphate (SDS) 5% w/v aqueous solution
Batch:1294323
Purity:unknown
Expiry Date:25 October 2017
Storage Conditions:room temperature

MATERIALS AND METHODS
Test System
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received: 08 October 2013
EpiSkinTM Tissues (0.38cm2)
lot number : 13-EKIN-035
Maintenance
Medium lot number:13-MAIN3-042
Assay Medium lot number : 13-ESSC-036
Test Item Formulation and Experimental Preparation
The test item was used as supplied.
Preparation of Negative and Positive Control Items, MTT and Acidified Isopropanol
The negative control item was used as supplied.
The positive control item was prepared as a 5% w/v aqueous solution.A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to0.3 mg/mL with assay medium when required.A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTTMTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.Test for Direct MTT ReductionAs specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:10 µ L 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.

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.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. 10 µ L of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µ L of DPBS served as the negative controls and triplicate tissues treated with 10 µ L of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particularcare to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.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 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 µ L 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 µ L samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µ L of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Interpretation of Results
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way: Relative mean visibility (%): (mean OD562 of test item / mean OD562 of negative control) x 100
Assay Acceptance Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Positive Control
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18%.Negative ControlThe assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues was 0.6, and the standard deviation value of the percentage viability is ≤ 18%.3.7.3
Test Item
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤ 18%.
Irritation / corrosion parameter:
other:
Value:
105.9
Remarks on result:
other:
Remarks:
Basis: other: relative mean viability of the test item treated tissues. Time point: 15 minutes. Reversibility: no data. (migrated information)
Irritant / corrosive response data:
RESULTS
Direct MTT Reduction
The MTT solution containing the test item turned remained yellow which indicated that the test item did not directly reduce MTT.
Test Item, Positive Control Item and Negative Control Item
The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.The relative mean viability of the test item treated tissues was 105.9% after a 15-Minute exposure period and 42 hours post-exposure incubation period.It was considered unnecessary to perform IL-1 analysis as the results of the MTT test were unequivocal.
Other effects:
Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 9.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.2%. The positive control acceptance criterion was therefore satisfied.The mean OD562 for the negative control treated tissues was 0.811 and the standard deviation value of the percentage viability was 5.7%. The negative control acceptance criterion was therefore satisfied.The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.2%. The test item acceptance criterion was therefore satisfied.

Table1          Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

  

Item

OD562of tissues

Mean OD562of triplicate tissues

±SDof OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.845

0.811

0.046

104.2

100*

5.7

0.829

102.2

0.758

93.5

Positive Control Item

0.056

0.076

0.018

6.9

9.4

2.2

0.090

11.1

0.082

10.1

Test Item

0.904

0.859

0.066

111.5

105.9

8.2

0.783

96.5

0.890

109.7

SD=        Standard deviation

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

Interpretation of results:
not irritating
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply: CLP: Not classified for Irritation. UN GHS Not classified for Irritation (category 3 cannot be determined).
Executive summary:

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
11 to 28 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with current OECD guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.35 to 2.73 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.1 mL of the test item
Duration of treatment / exposure:
7 days
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3
Details on study design:
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.Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered approximately 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Approximately 5 minutes prior to test item application, a pre-dose anaesthesia 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 similarly treated.Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977).Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.Any clinical signs of toxicity, if present, were also recorded.An additional observation was made in one treated eye on Day 7 to assess the reversibility of the ocular effects.Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.Interpretation of ResultsThe numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:Score for conjunctivae = (A + B + C) x 2Score for iris = D x 5Score for cornea = (E x F) x 5Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 72 hours
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 72 hours
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: 72 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: 48 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 24 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: 48 hours
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 24 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Ocular Reactions
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2 below.No corneal effects were noted during the study.Iridial inflammation was noted in one treated eye one hour after treatment. No effects were noted at 24 hours.Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation.Two treated eyes appeared normal at the 72-Hour observation and one treated eye appeared normal at the 7-Day observation.
Other effects:
Body Weight
Individual body weights and body weight change are given in Table 3 below.One animal showed expected gain in body weight during the study. No gain in body weight was noted in one animal and one animal showed body weight loss during the study.

Table1  Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

73717Male

73758Male

73759Male

IPR= 0

IPR = 0

IPR = 0

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

7
Days

1
Hour

24
Hours

48
Hours

72
Hours

1
Hour

24
Hours

48
Hours

72
Hours

CORNEA

 

 

 

 

 

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

 

 

 

 

D

1

0

0

0

0

0

0

0

0

0

0

0

0

Score (D x 5)

5

0

0

0

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

 

 

 

A = Redness

1

1

1

1

0

1

1

1

0

1

1

1

0

B = Chemosis

1

1

1

1

0

1

1

0

0

1

1

1

0

C = Discharge

1

0

0

0

0

1

1

0

0

1

1

0

0

Score (A + B + C) x 2

6

4

4

4

0

6

6

2

0

6

6

4

0

Total Score

11

4

4

4

0

6

6

2

0

6

6

4

0

 

Table2  Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number

and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

7 Days

73717Male

11

4

4

4

0

73758Male

6

6

2

0

-

73759Male

6

6

4

0

-

Group Total

23

16

10

0

0

Group Mean Score

7.7

5.3

3.3

1.3

0.0

 

Table3  Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

73717 Male

Day 0

Day 7

0.14

2.73

2.87

73758 Male

Day 0

Day 3

0.00

2.36

2.36

73759 Male

Day 0

Day 3

-0.01

2.35

2.34

IPR=Initial pain reaction

- =         Observation not required - considered to be zero for calculation of Group Mean Score

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a maximum group mean score of 7.7 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
Executive summary:

An assessment in vitro was not conducted. This is because OECD Guideline 438 is not a full test for eye irritation, and only provides information on whether a substance is a severe eye irritant. A negative finding would need to be followed by an in vivo study. Equivalent information on irritancy information can be obtained from the in-vitro skin corrosivity study. As this study was negative , severe eye irritation was not anticipated as the substance was not corrosive. It was considered appropriate to proceed directly to an in vivo test on these grounds.

The test item produced a maximum group mean score of 7.7 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

No classification is applicable.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The following data sets support the assessment of the substance as a “non-irritant”.

 

Skin Irritation

 

Evaluation under REACH Progress Report 2010, Pg 32 states as follows:

 

Skin irritation-corrosion

Annexes VIII-X requires an in vivo test to assess Skin irritation/corrosion. However, there are currently several in vitro methods available that can be used in a weight-of-evidence approach, to fully replace animal testing.

It is generally agreed that the EU B.46 (OECD 439) in vitro methods for Skin irritation represent a full replacement of the respective in vivo method (OECD 404) in a tiered testing strategy and in conjunction with in vitro skin corrosivity tests, if necessary. It should be noted that B.46 method does not address corrosivity; therefore, in case of positive result in a B46 test, a test addressing skin corrosion has to be performed.

 

It is recommended that the following testing strategy is followed when performing in vitro tests to assess skin-irritation and corrosion (see also Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance) - Skin corrosion shall be tested first; in case of positive results, no further testing is necessary; the substance shall be classified accordingly.

 

- If the results of the skin corrosion test is negative, then a skin irritation study according to EU method B.46 shall be performed; if the result is positive, no further testing is necessary but classification of the substance.

A negative result in the B.46 test does not need to be confirmed by additional testing.

 

Consequently, the default testing strategy would stop after testing skin irritation in vitro. No further in vivo testing would be required.

 

As the substance was negative in both in vitro screening studies, it is considered acceptable to waive the in vivo skin irritation study result on the above basis.

 

Eye Irritation

An assessment in vitro was not conducted. This is because OECD Guideline 438 is not a full test for eye irritation, and only provides information on whether a substance is a severe eye irritant. A negative finding would need to be followed by an in vivo study. Equivalent information on irritancy information can be obtained from the in-vitro skin corrosivity study. As this study was negative , severe eye irritation was not anticipated as the substance was not corrosive. It was considered appropriate to proceed directly to an in vivo test on these grounds.

No corneal effects were noted during the in vivo study. Iridial inflammation was noted in one treated eye one hour after treatment. No effects were noted at 24 hours.

Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation. Two treated eyes appeared normal at the 72-Hour observation and one treated eye appeared normal at the 7-Day observation.

  

Respiratory Irritation

No data is available for this endpoint; this is not a prescribed REACH endpoint. Historical use of white oils suggests that these materials are not respiratory irritants; the vapour pressure is not sufficiently high enough to cause effects. No effects are predicted.


Justification for selection of skin irritation / corrosion endpoint:
2 x in vitro tests available; both showing no effects.

Justification for selection of eye irritation endpoint:
1 x in vivo study available, showing no effects.

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Results of the studies are as follows:

Skin irritation.

The test item was classified as non-irritant and non-corrosive on the basis of two in vitro screening assessments. . The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 cannot be determined).

Justification is detailed above for this overall classification.

 

Eye Irritation.

The test item was classified as a mild irritant only on the basis of one in vivo assessment. The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation.

Justification is detailed above for this overall classification.