Registration Dossier

Administrative data

Description of key information

The skin irritation/corrosion potential of the test substance, Fret 10-0367 was assessed according to the following guideline studies:

OECD Test Guideline 439 using an in vitro skin irritation: Reconstructed Human Epidermis Model Test: This result was only just above the ≤ 50% threshold for triggering an irritant result and therefore it should be noted that even though this study was negative the test item does have the potential to cause a degree of response to the skin.

OECD Test Guideline 431 using a Reconstructed Human EpiDermis (RHE): The test item was considered to be non-corrosive to the skin.

OECD Test Guideline 404 using the Acute Dermal Irritation/Corrosion Method: The test item was classified as Category 3 (Mild irritant) according to the Globally Harmonized System of Classification and Labelling of Chemicals.No corrosive effects were noted. The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The eye irritation potential of the test substance, Fret 10-0367 was assessed according to the following guideline studies:

OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability (BCOP) Assay (2016): No prediction of eye irritation could be made.

OECD Test Guideline 492 using the In vitro Eye Irritation Test: Reconstructed Human EpiocularTM Model: The substance possesses an eye irritating potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 02 August 2016 and 05 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
(EC) No 440/2008, of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.
Cell source:
other: Not specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit supplied by MatTek
Source strain:
other: Not applicable
Details on animal used as source of test system:
Not applicable
Justification for test system used:
Recognised in vitro test for corrosivity
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 23348
- Delivery date: 02 August 2016
- Date of initiation of testing: 02 August 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37”C
- Temperature of post-treatment incubation: Room temperature.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: . Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL Sterile ditilled water
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8.0N Potassium Hudroxide
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 Minute exposure
Value:
ca. 127.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 Minute exposure
Value:
ca. 123.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of corrosion
Other effects / acceptance of results:
Quality Criteria
The mean OD562 for the negative control treated tissues was 1.656 for the 3 Minute exposure period and 1.596 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

The relative mean viabilities for each treatment group were as follows:

 Exposure Period  Percentage Viability       
   Negative Control  Positive Control  Test Item
 3 minute  100*  5.5  127.4
 60 minute 100*   5.4  123.3

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

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue.  Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control.  The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes.  Negative and positive control groups were treated for each exposure period.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.  After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 L samples were transferred to the appropriate wells of a pre-labeled 96 well plate.  The optical density (OD) was measured at 562 nm (OD562).

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

Results

The relative mean viabilities for each treatment group were as follows:

 Exposure Period  Percentage Viability       
   Negative Control  Positive Control  Test Item
 3 minute  100*  5.5  127.4
 60 minute 100*   5.4  123.3

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

Quality criteria:  

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

Conclusion

The test item was considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 16 February 2017 and 02 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 404 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch No. RDGM38757
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 21 June 2016
- Purity: 98.3%
- Appearance: Clear colorless liquid
- Storage condition of test material: Approximately 4”C in the dark
Cell source:
other:
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW
Details on test animals and environmental conditions:
Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.89 or 3.08 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 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.
Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, 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. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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.

Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
Test material:
Used as supplied
- Applied volume: 0.5 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:
4 Hours
Observation period:
1, 24, 48 and 72 hours
Number of animals:
2
Details on study design:
Study Design
On the day before the test each of a group of two rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.
Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in 74% industrial methylated spirit.
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation
preventing grading of erythema 4

Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and
extending beyond the area of exposure) 4

Any other skin reactions and clinical signs of toxicity, if present, were also recorded.
Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
animal: 1 and 2
Time point:
72 h
Score:
ca. 4
Max. score:
4
Reversibility:
not specified
Remarks on result:
probability of moderate irritation
Irritant / corrosive response data:
The test item produced a primary irritation index of 4.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.
The test item was classified as Category 3 (Mild irritant) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
Other effects:
Well defined erythema and slight edema were noted at one treated skin site with very slight erythema and slight edema noted at the other treated skin site immediately after patch removal. Well defined erythema and slight edema were noted at both treated skin sites 1 hour after patch removal and at the 24, 48 and 72 Hour observations.
Loss of skin elasticity was noted at both treated skin sites at the 24, 48 and 72 Hour observations with loss of skin flexibility also noted at both treated skin sites at the 72 Hour observation. Crust formation, which prevented accurate evaluation of erythema and edema, was noted at one treated skin site with moderate desquamation noted at the other treated skin site at the 7 Day observation.
Both treated skin sites appeared normal at the 14 Day observation.
No corrosive effects were noted.

Both animals showed expected gain in body weight during the study

Skin Reactions

The individual scores for erythema/eschar and edema are given in Table 1.

Well defined erythema and slight edema were noted at one treated skin site with very slight erythema and slight edema noted at the other treated skin site immediately after patch removal.  Well defined erythema and slight edema were noted at both treated skin sites 1 hour after patch removal and at the 24, 48 and 72 Hour observations.  

Loss of skin elasticity was noted at both treated skin sites at the 24, 48 and 72 Hour observations with loss of skin flexibility also noted at both treated skin sites at the 72 Hour observation.  Crust formation, which prevented accurate evaluation of erythema and edema, was noted at one treated skin site with moderate desquamation noted at the other treated skin site at the 7 Day observation.

Both treated skin sites appeared normal at the 14 Day observation.  

No corrosive effects were noted.

Body Weight

Both animals showed expected gain in body weight during the study.

Table 1: Individual Skin Reactions

 Skin Reaction

 Observation Time (following patch removal)

 Individual Scores   

 Total

 

 

 Rabbit 75698 Male

 Rabbit 75699 Male

 

 Erythema/Eschar Formation

 Immediately

 2

 1

 (3)

 

 1 Hour

 2

 2

 (4)

 

 24 Hours

 2Le

 2Le

 4

 

 48 Hours

 2Le

 2Le

 (4)

 

 72 Hours

 2LeLf

 2LeLf

 4

 

 7 Days

 0D

 ?eCf

 (0 -4)

 

 14 Days

 0

 0

 (0)

 Edema Formation

 Immediately

 2

 2

 (4)

 

 1 Hour

 2

 2

 (4)

 

 24 Hours

 2

 2

 4

 

 48 Hours

 2

 2

 (4)

 

 72 Hours

 2

 2

 4

 

 7 Days

 0

 ?od

 (0 -4)

 

 14 Days

 0

 0

 (0)

 Sum of 24 and 72 Hour Readings (S)       :       16

 Primary Irritation Index (S/4)              :       16/4 = 4.0

 Classification : MODERATE IRRITANT

(    ) =       Total values not used for calculation of primary irritation index

Cf =              Crust formation

D =              Moderate desquamation

Le =              Loss of skin elasticity

Lf =              Loss of skin flexibility

?e =              Adverse skin reaction prevented accurate evaluation of erythema

?od =       Adverse skin reaction prevented accurate evaluation of edema

Interpretation of results:
Category 3 (mild irritant) based on GHS criteria
Remarks:
according to the Globally Harmonized System of Classification and Labelling of Chemical
Conclusions:
The test item produced a primary irritation index of 4.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.
The test item was classified as Category 3 (Mild irritant) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

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

Results

A single 4 Hour, semi occluded application of the test item to the intact skin of two rabbits produced well-defined erythema and slight edema.  Other skin reactions noted were loss of skin elasticity and flexibility, moderate desquamation and crust formation (preventing accurate evaluation of erythema and edema).  Both treated skin sites appeared normal at the 14 Day observation.  No corrosive effects were noted.

Conclusion

The test item produced a primary irritation index of 4.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.  

The test item was classified as Category 3 (Mild irritant) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 21 September 2016 and 26 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
EC No. 761/2009 23 July 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch No. RDGM38757
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 21 June 2016
- Purity: 98.3%
- Appearance: Clear colorless liquid
- Storage condition of test material: Approximately 4”C in the dark
Cell source:
other:
Species:
other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model
Strain:
other: Not applicable
Details on test animals and environmental conditions:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 20 September 2016

EpiSkinTM Tissues (0.38cm2) lot number : 16-EKIN-038
Maintenance Medium lot number : 16-MAIN3-065
Assay Medium lot number : 16-ESSC-041
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
other:
Amount / concentration applied:
Test material:
Used as supplied
- Applied volume: 10 ul
- Concentration (if solution): undiluted

Positive control was prepared as a 5% w/v aqueous solution.
Duration of treatment / exposure:
15-Minute exposure period and 42 hours post-exposure incubation period.
Number of animals:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.
Details on study design:

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT 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 thecellular 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 by using killed tissues to act as controls.

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 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours.
Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarosegel and the insert:

Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

2.0 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of three 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):
2.0 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 μL of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL 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 particular care 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. Residual test item remained on the tissues after rinsing. The rinsed tissues were transferred to the second column of 3 wells containing 2.0 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.

2.0 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.
Irritation / corrosion parameter:
% tissue viability
Value:
50.6
Negative controls validity:
valid
Remarks:
Mean % viability = 100
Positive controls validity:
valid
Remarks:
Mean % viability = 10.1
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
This result was only just above the ≤ 50% threshold for triggering an irritant result and therefore it should be noted that even though this study was negative the test item does have the potential to cause a degree of response to the skin
Irritant / corrosive response data:
Dissiminated (delete when editing) add (average) score and conclude on irritancy (using CLP as standard)
Other effects:
Dissiminated (delete when editing): include when applicable

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple 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 the appendix.  The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in the appendix.

The relative mean viability of the test item treated tissues was 50.6% after a 15 Minute exposure period and 42 Hour post exposure incubation period.  This result was only just above the ≤ 50% threshold for triggering an irritant result.

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

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 10.1% relative to the negative control treated tissues and the standard deviation value of the viability was 1.1%.  The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.845 and the standard deviation value of the viability was 4.7%.  The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.2%.  The test item acceptance criterion was therefore satisfied.

Table: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD562

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

± SD of

Relative

mean

viability (%)

Negative

Control Item

0.820

0.845

0.040

97.0

100*

4.7

0.824

97.5

0.891

105.4

Positive Control Item

0.094

0.086

0.010

11.0

10.1

1.1

0.088

10.4

0.075

8.9

Test Item

0.434

0.428

0.018

51.4

50.6

2.2

0.442

52.3

0.407

48.2

SD = Standard deviation

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

Interpretation of results:
other: EU CLP Criteria Not classifiedfor irritation
Remarks:
UN GHS Not classified for Irritation (category 3 can not be determined).
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined).
This result was only just above the ≤ 50% threshold for triggering an irritant result and therefore it should be noted that even though this study was negative the test item does have the potential to cause a degree of response to the skin.
Executive summary:

The possible skin irritation potential of the substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 50.6%.

Since the mean relative tissue viability for FRET 10 -0367 was only just above the ≤ 50% threshold for triggering an irritant result and therefore it should be noted that even though this study was negative the test item does have the potential to cause a degree of response to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted on 31 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter,
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL as supplied
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Study Design
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96 well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Irritation parameter:
in vitro irritation score
Value:
ca. 7.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Corneal Opacity and Permeability Measurement
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

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

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore satisfied

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

 Treatment 1  Cornea Number  Opacity              Permeability (OD)     In vitro Irritancy score
     Pre-treatment  Post-treatment  Post incubation  Post incubation - pre-treatment  Corrected value    Corrected value  
 Negative contrrol  11  4  4  5  1    0.020    
   12  4  4  6  2    0.020    
   13  4  4  6  2    0.019    
           1.7*    0.020†    2.0
 Positive control  10  3  26  29  26  24.3  1.089  1.069  
   14  2  30  37  35  33.3  1.217  1.197  
   15  4  36  37  33  31.3  0.925  0.905  
             29.7**    1.057**  45.5
 Test Item  16  4  6  10  6  4.3  0.097  0.077  
   17  3  6  12  9  7.3  0.102  0.082  
   18  4  6  13  9  7.3  0.117  0.097  
             6.3**    0.086**  7.6

OD = Optical Density * = Mean of post-incubation - pre-treatment values       †= Mean premeability       **= Mean corrected value

Interpretation of results:
other: No prediction of eye irritation can be made
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage.  The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro.  In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes.  Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1.  Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

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

Data Interpretation

The test item is classified according to the prediction model as follows:

 IVIS  Classification
 ≤ 3  No category. Not requiring classification to UN GHS or EU CLP
 > 3; ≤55  No prediction of eye irritation can be made
 > 55 Category 1. UN GHS or EU CLP Causes serious eye damage 

Results

The In Vitro irritancy scores are summarized as follows:

 Treatment  In Vitro Irritancy Score
 Test Item  7.6
 Negative Control  2.0
 Positive Control  45.5

Conclusion

No prediction of eye irritation can be made.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 06 September 2016 and 08 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50µL dispensed directly on to EpiOcular tissue surface
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Duplicates
Details on study design:
Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsingthe tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for nearly 120 minutes at standard culture conditions (post-treatment incubation).

MTT Assay
After post-treatment incubation of 120 minutes the MTT assay was performed.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative, and positive controls.
Irritation parameter:
other: Mean Relative Absorbance
Remarks:
(% of negative control)
Run / experiment:
1
Value:
ca. 34.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% Negative control
Positive controls validity:
valid
Remarks:
28.9% Negative control
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
This in vitro study was performed to assess the eye irritation potential of Fret 10-0367 by means of the Human Cornea Model Test.
Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.
Tissues of the human cornea model EpiOcular™ were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.
50 μL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size.
Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 28.9%, thus the validity of the test system is ensured.
Relevant irritating effects were observed following 30 minutes incubation with Fret 10-0367. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 34.8% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

Results after treatment for 30 minutes with Fret 10-0367 and the controls:

 Dose Group

 Absorbance Well 1 (tissue 1/2)

Absorbance Well 2 (Tissue 1/2)

 Mean Absorbance (Tissue 1/2)

 Mean Absorbance* Tissue 1 and 2

 Mean Absorbance of 2 Tissues*

 Relative Absorbance (%) Tissue 1 and 2

 Absolute Valuse of Differnece of Re. Absorbacne (%) Tissue 1 and 2

 Mean Absorbacne % Negative control)**

 Blank

 0.039

 0.039

 0.039

 0.000

 

 

 

 

 Negative Control

 1.521

 1.625

 1.573

 1.534

 1.417

 108.3

 16.5

 100.0

 

 1.334

 1.344

 1.339

 1.300

 

 91.7

 

 

 Positive Control

 0.383

 0.377

 0.380

 0.341  0.409  24.1  9.6  28.9
   0.519  0.515  0.517  0.478    33.7    
 Test Item  0.490  0.475  0.482  0.443  0.493  31.3  7.1  34.8
   0.588  0.577  0.582  0.543    38.4       

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values) = (100x (absorbance test item/positive control)/(mean absorbancenegative control))

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. An

additional test with viable tissues without MTT additions was not necessary.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. An additional test with freeze-killed tissues did not have to be performed.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 34.8% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.

Concerning acceptance criteria:

• The negative control OD is > 0.8 and < 2.5 (values between 1.334 and 1.625).

• The mean relative viability of the positive control is below 50% of the negative control viability (28.9%).

• The difference of viability between the two relating tissues of a single item is < 20% (values between 7.1% and 16.5%) in the same run (for positive and negative control tissues and tissues of single test items).

Interpretation of results:
other: Fret 10-0367 possesses an eye irritating potential
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Fret 10-0367 possesses an eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of Fret 10-0367 by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues (without MTT addition) to gain correction factors for true viability calculation in the main experiment did not have to be performed.

Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with Fret 10-0367. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (34.8%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, Fret 10-0367 possesses an eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification

Based on the results in the in vivo skin irritation study (OECD TG 404), the substance does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and its amendments.

Based on the results in eye irritation tests (OECD TG 437 and 492), the substance is classified as an eye irritant caterogy 2 and shall be labelled as H319 "Causes serious eye irritation" according to Regulation (EC) No. 1272/2008 and its amendments.