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Description of key information

In vitro eye and dermal studies on polyglycerine have been conducted.

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:
20-26 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not specified
GLP compliance:
yes
Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
EPISKIN™ MODEL KIT was obtained from Skin Ethic Laboratories, Lyon, France.
Type of coverage:
open
Preparation of test site:
other:
Vehicle:
unchanged (no vehicle)
Controls:
other: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control. An aqueous solution of Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.
Amount / concentration applied:
10 uL
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
Tests were run in triplicate
Details on study design:
MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium 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 Reduction
As 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 uI 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.

The test item was shown to directly reduce MTT in the direct MTT reduction test (section 8.1: Direct MTT Reduction). There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues. Water-killed tissues were prepared by placing untreated EPISKIN™ tissues in a 12-well plate containing 2.2 ml of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO2 in air for 48 ±1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30°C) for up to 6 months. Before use each tissue was thawed by placing in 2.2 ml of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, each MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

Pre-Incubation (Day 0: tissue arrival)
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 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 uI of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 uI of OPBS served as the negative controls and triplicate tissues treated with 10 uI of SOS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SOS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SOS solution was re-spread with a pipette tip to maintain the distribution of the SOS 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 OPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of OPBS 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 homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C 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 MIT 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% C02 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 EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 uI 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 MIT-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 coloured solution.

For each tissue, duplicate 200 uI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 uI of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.
Irritation / corrosion parameter:
other: other: relative tissue viability
Value:
95
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hour. Reversibility: no data. (migrated information)
Irritant / corrosive response data:
Direct MTT Reduction
The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT.

The direct reduction by the test item relative to the negative control value was 6.2%

Direct reduction was therefore acceptable.

Test Item, Positive Control Item and Negative Control Item
The individual and mean OD540 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 95.0% after a 15-Minute exposure period.
Other effects:
No additional information available

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.3%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.744 and the standard deviation value of the percentage viability was 11.6%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 3.1 %. The test item acceptance criterion was therefore satisfied.

Table 1

DETERMINATION OF SKIN IRRITATION POTENTIAL USING THE EPISKIN™ RECONSTRUCTED

HUMAN EPIDERMIS MODEL

Mean OD540 Values and Percentage Viabilities for Control Item, Positive Control Item and Test Item

 Material  OD540 of tissues tvt  OD540 of tissues corrected for direct reduction of MTT (0.046)  Mean OD540 (SD) Relative individual tissue viability (%)    Relative mean viability (%) (SD)
 Negative Control  0.751    0.744 (0.086)  100.9  100 (11.6)*
   0.655      88.0  
   0.827      111.2  
 Positive Control  0.045    0.062 (0.017)  6.0  8.3 (2.3)
   0.061      8.2  
   0.079      10.6  
 Test Item  0.727  0.681  0.707 (0.023)  91.5  95.0 (3.1)
   0.759  0.713    95.8  
   0.772  0.726    97.6  

Corrected viability of treated killed tissues = 0.130 (tkt) - 0.084 (ukt) = 0.046

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

OD540 = optical density

SD = Standard deviation

tvt = treated viable tissues

tkt = treated killed tissues

ukt = untreated killed tissues

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be Non-Irritant (NI).
Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric 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 is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will

be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

• OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010)

• Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: 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 postexposure incubation period each tissue was taken for MTT-Ioading. The maintenance medium from beneath each tissue was transferred to pre-labelled 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-labelled 96-well plate. The optical density was measured at 540 nm.

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

Results: The relative mean viability of the test item treated tissues was 95.0% after the 15-Minute exposure period.

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

Conclusion: The test item was considered to be Non-Irritant (NI).

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:
weight of evidence
Study period:
29-31 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Draft guideline is being considered.
Qualifier:
no guideline available
Principles of method if other than guideline:
The SkinEthic HCE model consists of transformed human corneal epithelial cells of the cell line HCE (LSU EYE center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test item is applied directly to the culture surface, at the air interface so that undiluted material can be tested directly.
GLP compliance:
yes
Species:
human
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Skinethic HCE was obtained from SkinEthic Laboratories, Lyon, France on 29 January 2013. On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated 'arrival plates'containing 300 ul of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37C, 5% CO2 in air.
Vehicle:
unchanged (no vehicle)
Controls:
other: yes, Solution A was used which consisted of Na2HPO4 0.142 g/L, glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L.
Amount / concentration applied:
30 ul
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
incubated for 3 hours after treatment and then allowed to remain at room temperature to extract MTT from tissue
Number of animals or in vitro replicates:
Each test was run in triplicate
Details on study design:
Preparation of Tissues
Using sterile techniques, 1 ml of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated 'treatment plates'. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7-Day old tissues were transferred from the 'arrival plates' into the wells of the 'treatment plates' containing the maintenance medium.

Main Test
Triplicate tissues were treated with 30 ul of the test item for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 ul of solution A to serve as negative controls and triplicate tissues were treated with 30 ul of 2% w/v SDS to serve as positive controls. The plates were incubated at 37C, 5% CO2 in air during the exposure time.

At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated 'holding plate' containing 300 ul of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per plate) were transferred to a pre-labelled 24-well plate designated 'MTT Loading plate' containing 300 ul of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37C, 5% CO2 in air.

At the end of the incubation period the inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated 'MTT extraction plate' containing 0.75 ml of isopropanol in each of a sufficient number of wells. An extra 0.75 ml of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminum foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 ul tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 ul samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 ul of isopropanol alone was added to three wells designated as 'blanks'. The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using the Anthos 2001 microplate reader.

Tissue Histology
One tissue for each treatment group was retained for possible tissue histopathology.

The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.

Interpretation of Results
The mean OD540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader.

The relative mean tissue viability (percentage of the negative control was calculated as follows:

Relative mean tissue viability (%) = [(mean LD540 of test item)/(mean OD540 of negative control)] x 100

When a test item was shown to directly reduce MTT, and therefore freeze-killed tissues were employed, the results of the MTT assay are corrected as follows:

True viability = mean OD tvt -(OD tkt -OD ukt)

OD = optical density at 540 nm
tvt = treated viabile tissues
tkt = treated killed tissues
ukt = untreated killed tissues

If the direct reduction by the test item was greater than 30% of the negative control value, additional steps must be takin into account, or the test item was considered to be incompatible with this test system.

If the direct reduction of MTT by the test item was less than 30% of the negative control value, the net OD of the test item treated killed control will be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

The mean tissue viability for the test item was compared to the negative control and classified to Table 1 below.

Assay Acceptance Criterion
The results of the assay are considered acceptable if the following assay acceptance criterion was achieved:

Assay Acceptance Criterion: Positive Control
The assay meets the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is <60% relative to the negative control treated tissues.
Irritation parameter:
in vitro irritation score
Value:
7.9
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
44.7
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Assessment of Direct Test Item Reduction of MTT
The MTT solution containing the test item turned blue which indicated that the test time directly reduced MTT and therefore the MTT viability assay was performed in parallel on viable and freeze-killed tissues. However, the results obtained showed no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Eye Irritation Potential
The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 7.9% (Table 1).

It was considered unnecessary to proceed with tissue histopathology.

Assay Acceptance Criterion
The quality criterion required for the acceptance of results in the test was satisfied.

Table 2 Assessment of Eye Irritation Potential - Viability of HCE Tissues

 Item  Mean OD540  Relative Mean Viability (%)
 Negative Controls  0.942  100*
 Positive Control  0.421  44.7
 Test Item  0.074  7.9

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

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
According to the study plan followed the test item was considered to be an irritant (I).
Executive summary:

Introduction. The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. The experimental design of the study consists of a test for direct reduction of MTT (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 ul of the test item for 10 minutes. Triplicate tissues treated with 30 ul of Solution A served as the negative control and triplicate tissues treated with 30 ul of 2% w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Date are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

i) If the percentage relative mean tissue viability was >60% the test item was considered to be non-irritant (NI).

ii) If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant (I).

Results. The relative mean viability of the test item treated tissues after a 10 -Minute exposure period was 7.9%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criterion. The quality criterion required for acceptance of results in the test was satisfied.

Conclusion. According to the study plan followed the test item was considered to be an Irritant (I).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

There are no in vivo dermal or eye irritation studies on polyglycerine. In vitro studies have been conductedaccording to REACh requirements to decease animal testing and using validated in vitro methods to estimate hazards.. In an Episkin study with 2% caustic, polyglycerine was not corrosive to the skin nor was it found to be an irritant. Similarly, the test material was considered not to be an ocular corrosive or severe irritant in the Bovine Corneal Opacity and Permeability (BCOP) assay. However in the Human Corneal Epithelium (HCE) assay, the material was found to be an irritant. Given that up to 2% NaOH may be present in the test material, the material is considered to be moderately irritating to the eye or skin. Thus based on the HCE assay results and the presence of up to 2% NaOH possible in the test material, this material is considered a Cat 2 for the eye.


Justification for selection of skin irritation / corrosion endpoint:
Polyglycerine is considered to be Non-Irritating (NI).

Justification for selection of eye irritation endpoint:
In the in vitro Human Corneal Epithelium assay, the test material containing 2% NaOH was considered to be an irritant.

Effects on eye irritation: moderately irritating

Justification for classification or non-classification

There is no justification for classification on skin based on data from available studies.

For eye, given the in vitro test results, polyglycerine is a Cat 2.