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Description of key information

Skin irritation:  An OECD 439 (Determination of Skin Irritation Potential using the EpiSkin Reconstructed Human Epidermis Model) study has been performed under the conditions of GLP. The Klimisch reliability is 1. 
Eye irritation: An in vivo study was performed on the test substance. The study was conclusive, performed to a valid guideline (OECD TG 420, adopted 17 December 2001) and was conducted under GLP conditions. The Klimisch reliability is 1.

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:
From 13 January 2015 to 19 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conclusive, performed to a valid guideline (OECD TG 439, adopted 22 July 2010) and was conducted under GLP conditions. No deviations from the test methods were noted.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP Inspection: 17 June 2015 Date of Signature on Certificate: 24 September 2015
Species:
other: reconstructed human epidermis
Strain:
other: reconstructed human epidermis
Details on test animals or test system and environmental conditions:
EpiSkin™ Reconstructed Human Epidermis Model Kit

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.

Supplier : SkinEthic Laboratories, Lyon, France
Date received : 13 January 2015
EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-002
Maintenance Medium lot number: 15-MAIN3-002
Assay Medium lot number : 15-ESSC-002

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

Adaptation to cell culture conditions (pre-incubation): 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.

Incubation Conditions:
37 °C, 5% CO2 in air overnight.
Type of coverage:
other: open in vitro system
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
water
Controls:
other: Positive and negative control items were used
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2)

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 µL of sterile distilled ater was topically applied to the epidermal surface prior to applying the tst item in order to improve contact between the test item and the epidermis

NEGATIVE CONTROL ITEM:

Identification: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Batch: 1553513
Purity: Not supplied
Expiry Date: 01 March 2017
Storage Conditions: Approximately 4°C in the dark

POSITIVE CONTROL ITEM:
Identification: Sodium Dodecyl Sulphate (SDS)
Batch: 1294323
Purity: Not supplied
Expiry Date: 25 October 2017
Storage Conditions: Room temperature

The negative control item, DPBS, was used as supplied.

The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
Duration of treatment / exposure:
15 minutes
Observation period:
Not applicable. The tissues were transferred to the second column of 3 wells containign 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Number of animals:
Not applicable. The test was performed in triplicates for each treatment and control group.
Details on study design:
PREPARATION OF MTT AND ACIDIFIED ISOPROPANOL

A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.

A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

REMOVAL OF TEST SUBSTANCE
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.

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT

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.

To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 mg 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.

MTT LOADING/FORMAZAN EXTRACTION (DAY 3 OF MAIN TEST)

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

INTERRETATION 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 viability (%) = (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 Control
The 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.

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: other: relative mean tissue viability (%)
Value:
114.1
Remarks on result:
other:
Remarks:
Basis: other: mean value test item. Time point: 15 minute exposure. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: relative mean tissue viability (%)
Value:
28.6
Remarks on result:
other:
Remarks:
Basis: other: mean value positive control. Time point: 15 minute exposure. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: relative mean tissue viability
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: mean value positive control item. Time point: 15 minute exposure. Max. score: 100.0. Reversibility: other: not applicable. Remarks: The mean viability of the negative control tissues is set at 100%. (migrated information)
Irritant / corrosive response data:
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 below. 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 114.1% after a 15-Minute exposure period and 42 hours post-exposure incubation period.

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Table 1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive

Control Item and Test Item

Item

OD562of tissues

Mean OD562of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean tissue viability (%)

± SD of Relative mean viability (%)

Negative control item

0.677

 

0.675

 

0.02

100.3

 

100*

 

3.5

0.697

103.3

0.651

96.4

Positive control item

0.207

 

0.193

 

0.02

 

30.7

 

28.6

 

 

3.5

0.166

24.6

0.206

30.5

 

Test item

0.770

 

0.770

 

 

0.05

114.1

 

114.1

 

 

7.7

0.718

106.4

0.822

121.8

 

SD = Standard deviation

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

OD = Optical density

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.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 28.6% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 3.5%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.675 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 7.7%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was classified as non-irritant.
Executive summary:

Introduction

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. 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 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.

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 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).

Results

The relative mean viability of the test item treated tissues was 114.1% after the 15-Minute exposure period and 42 hours post-exposure incubation 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.

Therefore, classification according to EU DSD (Council Directive 67/548/EEC), EU CLP (Regulation (EC) No 1272/2008) and UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is not required.

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:
From 09 March 2015 to 30 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conclusive, performed to a valid guideline (OECD TG 405, adopted 2 October 2012) and was conducted under GLP conditions. No deviations from the test methods were noted.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 12 to 14 March 2015 Date of Signature on Certificate: 12 May 2015
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratoires UK Ltd., Leicestershire, UK
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.95 or 3.12 kg
- Housing: Individually in suspended cages
- Diet (e.g. ad libitum): 2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK was supplied ad libitum
- Water (e.g. ad libitum): Mains drinking water provided ad libitum.
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): At least fifteen per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye of each animal remained untreated and was used for control purposes.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL, approx 84 mg (as measured by gently compacting the required volume into an adapted syringe)


Observation period (in vivo):
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.
Number of animals or in vitro replicates:
Two - after consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Details on study design:
ANALGESIA
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.

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.

TEST PROCEDURE
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. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): None

SCORING SYSTEM: Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment according to the Draize scale (Appendix 2).

TOOL USED TO ASSESS SCORE: Standard opthalmascope
Irritation parameter:
cornea opacity score
Basis:
animal: 74927 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal: 75004
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal: 74927 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.33
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Basis:
animal: 75004 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.33
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: redness
Basis:
animal: 74927 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
other: redness
Basis:
animal: 75004 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: 74927 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: 75004 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Individual scores for ocular irritation are given in Table 1. Individual and Mean Scores for Cornea, Iris and Conjunctivae required for classification and labelling are given in Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in both treated eyes 1 and 24 hours after treatment.
Moderate conjunctival irritation was noted in all treated eyes 1 hour after treatment and at the 24 and 48-Hour observations with minimal conjunctival irritation noted at the 72-Hour observation.
Both treated eyes appeared normal at the 7-Day observation.
Other effects:
Individual body weights and body weight change are given in Table 3.
Both animals showed expected gain in body weight during the study.

Table 1: Individual Scores for Ocular Irritation

Rabbit Number and Sex

74927Male

75004 Male

Initial Pain Response = 0

Initial Pain Response = 0

Time After Treatment

1 Hour

24 Hours

48 Hours

72 Hours

7 Days

1 Hour

24 Hours

48 Hours

72 Hours

7 days

CORNEA

 

 

 

 

 

 

 

 

 

 

Degree of Opacity

0

0

0

0

0

0

0

0

0

0

Area of Cornea Involved

0

0

0

0

0

0

0

0

0

0

IRIS

1

1

0

0

0

1

1

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

Redness

2

2

2

2

0

2

2

2

2

0

Chemosis

2

2

2

1

0

2

2

2

1

0

Discharge

1

1

0

0

0

2

2

1

0

0

Table 2 Individual and Mean Scores for Cornea, Iris and Conjunctivae

Rabbit Number and Sex

Time After Treatment

Corneal Opacity

Iridial Inflammation

Conjuctival Redness

Conjunctival Chemosis

 

74927 Male

24 Hours

0

1

2

2

48 Hours

0

0

2

2

72 Hours

0

0

2

1

Mean

0.0

0.3

2.0

1.7

 

75004 Male

24 Hours

0

1

2

2

48 Hours

0

0

2

2

72 Hours

0

0

2

1

Mean

0.0

0.3

2.0

1.7

Table 3 Individual Bodyweights and Bodyweight Change

Table 3 Individual Bodyweights and Bodyweight Change

Rabbit Number and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 7

74927 Male

3.12

3.18

0.06

75004 Male

2.95

3.00

0.05

 
Interpretation of results:
Category II
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced individual scores of 0.0 for corneal opacity, 0.3 for iritis, 2.0 for conjunctival redness and 1.7 for chemosis, calculated as the mean scores following grading at 24, 48 and 72 hours after instillation. Observed effects were fully reversible within the observation period.

The test item was classified as Category 2B (mildly irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 7-Day observation.

Conclusion

The test item produced individual scores of 0.0 for corneal opacity, 0.3 for iritis, 2.0 for conjunctival redness and 1.7 for chemosis, calculated as the mean scores following grading at 24, 48 and 72 hours after instillation. Observed effects were fully reversible within the observation period. The test item was classified as Category 2B (mildly irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
An OECD 439 (Determination of Skin Irritation Potential using the EpiSkin Reconstructed Human Epidermis Model) study has been performed under the conditions of GLP. This study is considered to be the key data for classification and labelling. A negative in vitro corrosion study is also provided.

Justification for selection of eye irritation endpoint:
One key study available.

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation: An OECD 439 (Determination of Skin Irritation Potential using the EpiSkin Reconstructed Human Epidermis Model) study has been performed under the conditions of GLP. The result was negative and therefore as the study is considered reliable for classification and labelling in accordance with Regulation (EC) No. 1907/2008 the test material is considered to be non-classified.

Eye irritation: The test item produced individual scores of 0.0 for corneal opacity, 0.3 for iritis, 2.0 for conjunctival redness and 1.7 for chemosis, calculated as the mean scores following grading at 24, 48 and 72 hours after instillation. Observed effects were fully reversible within the observation period. Ammonium polyphosphate is classified as category 2, irritating to the eyes in accordance with Regulation (EC) No.1272/2008 (EU CLP).