Registration Dossier

Administrative data

Description of key information

Skin: Based on the two in vitro skin irritation/corrosion studies, it was concluded that the test substance is non corrosive and a non iirrtant to the skin. A key invivo study on one of the major constituents of the test item as well as a supporting in vivo study concluded that the test substance is non-irritating to the skin

Eye: Based on an in vitro eye irritation test (BCOP) results, the test substance is not classified for eye irritation or serious eye damage. In another invitro study, "PP-R-001 (CAS#1613243-54-1): IN VITRO EYE IRRITATION TEST USING HUMAN CORNEA MODEL (EpiOcular™ Eye Irritation Test)" it was concluded that under this OECD 492 GLP study conditions, CAS#1613243-54-1 does not possess any eye irritating potential.

Additionally, a key in vivo study and a supporting an in vivo study on CAS# 24748-23-0 (one of the major constituents of CAS# 1613243-54-1, the test item), concluded that it would not be classified as an eye irritant in accordance with the criteria for classification in accordance with the Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 Nov, 2012.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April-May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study acording to GLP
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Three New Zealand White rabbits supplied by David Percival Ltd, Moston,
Sandbach, Cheshire, UK were used. At the start of the study the animals
weighed 2.61 to 2.71 kg and were twelve to sixteen weeks old. After a
minimum acclimatisation period of five days each animal was given a number
unique within the study which was written with a black indelible marker-pen
on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended metal cages. Free access
to mains drinking water and food (STANRAB SQC Rabbit Diet, Special Diets
Services Ltd, Witham, Essex, UK) was allowed throughout the study.

The animal room was maintained at a temperature of 17 to 21 . C and relative
humidity of 46 to 65%. The rate of air exchange was approximately fifteen
changes per hour and the lighting was controlled by a time switch to give
twelve hours continuous light and twelve hours darkness.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: None
Controls:
no
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
4 h
Observation period:
1, 24, 48, 72 h up to 14 days
Number of animals:
3
Details on study design:
On the day before the test each rabbit 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 material was introduced under a 2.5 cm
x 2.5 cm cotton gauze patch and placed in position on the shorn skin. The
patch was secured in position with a strip of surgical adhesive tape
(BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals
interfering with the patches, the trunk of each rabbit was wrapped in an
eJasticated corset (TUBIGRJP) 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 material removed by gentle swabbing with cotton
wool soaked in 74% Industrial Methylated Spirits.

Approximately one hour following the removal of the patches, and 24, 48 and
72 hours later, the test sites were examined for evidence of primary irritation
and scored according to the following scale from Draize J H, (1977) "Dermal
and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the
Toxicity of Household Substances, National Academy of Sciences, Washington
DC p.31.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24, 48 and 72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Reversibility of any observed effect: Changes fully reversible within 14 days
Other effects:
None

Summary of dermal lesions (following 4-h application)

 

Animal no.

Effect

Hour

Days after application

Mean score erythema

(24/48/72 h)

Mean score oedema

(24/48/72 h)

1

1

2

3

7

14

115

Erythema

Oedema

2

1

2

1

2

1

2LeLf

1

0D

0

0

0

2

 

1

59

Erythema

Oedema

1

0

2

1

2

1

2

1

1D*

0

0

0

2

 

1

142

Erythema

Oedema

2

1

2

1

2

1

2

1

0D

0

0

0

2

 

1

Mean all animals

 

 

 

 

 

 

 

 

2

 

1

 

Le= loss of skin elasticity, Lf= loss of skin flexibility, D=slight desquamation, D*= moderate desquamation

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on a mean erythema score of 2 and a mean oedema score of 1 and full reversibility of the effects within 14 days, the test substance does not meet the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classifica tion, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012. However, the test material would be classified as R 38 in accordance with Annex VI of Council Directive 67/548/EEC, Relating to the Classification, Packaging and Labelling of Dangerous Substances.
Executive summary:

The study was performed to assess the irritancy potential of the test material following a single, 4-hour, semi-occluded application to the intact rabbit skin (Safepharm Standard Method Number 540.01). The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 17 July 1992) and Method B4 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Three New Zealand White rabbits supplied by David Percival Ltd, Moston, Sandbach, Cheshire, UK were used. On the day before the test each rabbit 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 material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape (BLENDERM: approximate size 2.5 cm x 4.0 cm). To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an eJasticated corset (TUBIGRJP) 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 material removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated Spirits.

Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J H, (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.31.

The test material produced positive criteria in 3/3 rabbits according to the EU labelling regulations, Annex VI of CounciDirective 67/548/EEC, Relating to the Classification, Packaging and Labelling of Dangerous Substances, and was classified as IRRITANT to rabbit skin (R 38 "IRRITATING TO SKIN"). However, based on a mean erythema score of 2 and a mean oedema score of 1 and full reversibility of the effects within 14 days, the test substance does not meet the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental stat date: 04/11/14 Experimental completion date: 06/11/14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EPIDERM (TM) Reconstructed Human Epidermis Model Kit
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Supplier : MatTek Corporation, Ashland, MA, USA
Date received : 04 November 2014
EpiDermTM Tissues (0.5cm2) lot number it K
Assay Medium lot number : 103014ZSD

Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator overnight.

Type of coverage:
other: Test item applied to culture surface, at air interface
Preparation of test site:
other: The assay medium was pre-warmed before use. 0.9 mL of assay medium was pipetted into the appropriate wells of 2 pre-labeled 6-well plates for both the 3 Min, and 60 Min, exposure periods. EpiDerm tissues were transferred into the 6 well plate
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative Control: Sterile Distilled Water Positive Control: 8.0N Potassium Hydroxide
Amount / concentration applied:
Amount applied: 50 µL per well

Concentration: not stated in study report4
Duration of treatment / exposure:
3 minutes and 60 minutes
Observation period:
Immediate observation following exposuremn
Number of animals:
Two x 24-well plates
Details on study design:
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Dye 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 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 was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:

100 L of the test item was added to 900 L of a 1.0 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. 100 L of sterile distilled water was tested concurrently to act as a control.

If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.


Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.

After pre incubation of the EpiDerm tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.

When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.

After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (Isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the blue formazan extract was transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
79.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: reversibility not determined
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
87.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: reversibility not determined
Other effects / acceptance of results:
The relative mean viability of the test item treated tissues was as follows:

60 minutes exposure : 79.3%
3 minutes exposure : 87.1%

Direct MTT Reduction
The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

TABLE

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

Item

Exposure Time

Mean OD5621

% Viability

Negative Control

3 minute

2.389

100*

60 minute

2.417

100*

Positive Control

3 minute

0.123

5.1

60 minute

0.054

2.2

Test Item

3 minute

2.080

87.1

60 minute

1.917

79.3

 

 


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

1=   Mean of EpiDerm tissues tested in duplicate

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 5.1 % relative to the negative control treated tissues following the 3‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562for the negative control treated tissues was 2.389 for the 3‑Minute exposure period and 2.417 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:
other: non-corrosive
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
The test item, PP-R-001 (CAS#1613243-54-1), was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item, PP-R-001 (CAS#1613243-54-1), 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. 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 mL 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 of the test item treated tissues were:

 

60 minutes exposure

:

79.3%

3 minutes exposure

:

87.1%

 

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

  

Conclusion

The test item, PP-R-001 (CAS#1613243-54-1), was considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: June 17th 2015 Experimental completion date: June 29th 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results..
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EPISKIN TM reconstructed human epidermis model
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 23 June 2015
EpiSkinTM Tissues (0.38cm2) lot number 15-EKIN025
Maintenance Medium lot number :15-MAIN3-025
Assay Medium lot number : 15-ESSC-025
Type of coverage:
other: . The test item was applied topically to the corresponding tissues ensuring uniform covering.
Preparation of test site:
other: Please see below for "Details on study design"
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: DPBS Positive control: SDS
Amount / concentration applied:
10 µL (26.3 µL /cm2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
15 minutes
Observation period:
Observations after 42 hour's incubation at 37°C, 5% CO2 in air then observations taken Day 6.
Number of animals:
12 well plate for each of the following groups: Test item, negative control, positive control
Details on study design:
Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

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

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.


Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 C, was pipetted into the second column of 3 wells of the 12 well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL /cm2) 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 a 7 Minute 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 (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. 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.


3.5.3 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:
other:
Value:
54.1
Remarks on result:
other:
Remarks:
Basis: other: Tissue viability of wells in each exposure group. Time point: 15 minute exposure period plus 42-hour post-exposure incubation period. Reversibility: other: not determined. Remarks: Result expressed as a percentage. (migrated information)
Other effects / acceptance of results:
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.
The relative mean viability of the test item treated tissues was 54.1% after a 15 Minute exposure period and 42 Hour post exposure incubation period.

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

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

Item

OD562of tissues

Mean OD562of triplicate tissues

±SDof OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.848

0.873

0.026

97.1

100*

3.0

0.899

103.0

0.872

99.9

Positive Control Item

0.086

0.079

0.009

9.9

9.1

1.1

0.069

7.9

0.083

9.5

Test Item

0.483

0.472

0.015

55.7

54.1

1.8

0.474

54.3

0.456

52.2

 

OD= Optical Density

SD=        Standard deviation

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

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 9.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562for the negative control treated tissues was 0.873 and the standard deviation value of the percentage viability was 3.0%. 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 1.8%. 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, PP R 001 (CAS#1613243 54 1), was classified as non irritant. The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item, PP-R-001(CAS#1613243-54-1),using the EPISKINTMreconstructed 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 54.1% 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, PP-R-001 (CAS#1613243-54-1), was classified as non-irritant. The following classification criteria apply:

 

EU DSD and CLP Not classified for Irritation.

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

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted GLP study. No certificate of analysis included in the report.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Albino Rabbit, New Zealand White, (SPF-Quality)
Source: Broekman Institute, Someren, The Netherlands.


Air-conditioned room with approximately 15 air changes per hour and the
environment controlled with optimal conditions considered as being a temperature
of 21°C and a relative humidity of 50%. Fluctuations from these optimal
conditions were noted, but were considered not to have affected study integrity.
Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Accommodation
Individually in labelled cages with perforated floors (Scanbur Denmark) and
equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
Acclimatisation period was at least 5 days before start of treatment under
laboratory conditions.

Diet
Standard laboratory rabbit diet (LKK-20, pellet diameter 4mm, Hope Farms,
Woerden, The Netherlands) approx. 100 gram per day. Certificates of analysis
were examined and retained in the NOTDX archives.
In addition, hay (BMI, Helmond, The Netherlands) was provided once a week.

Water
Free access to tap-water diluted with decalcified water.
Certificates of quarterly analysis were examined and retained in the NDTDX
archives.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.5 ml
Duration of treatment / exposure:
4 hours
Details on study design:
Approximately 24 hours before treatment, the dorsal fur was clipped with
electric clippers, exposing an area of approximately 150 square centimeters
(10x15 cm').
A health inspection was performed prior to the commencement of treatment, to
ensure that the animals were in a good state of health. Special attention was
paid to the skin to be treated, which was intact and free from abnormalities.
On test day 1, 0.5 ml of the test substance was applied to the skin of one
flank, using a surgical gauze patch of 2x3 cm. The patch was mounted on
Micropore tape*. which was wrapped around the abdomen and secured with Coban
elastic bandage.
Four hours after the application, the dressing was removed and the remaining
test substance removed using a tissue moistened with tap-water and subsequently
a dry tissue.
Whenever considered necessary the treated skin areas were re-clipped at least 3
hours before the observations, to facilitate the scoring .
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 24 and 72 hours
Score:
3.7
Max. score:
2.3
Reversibility:
fully reversible within: 14 days
Irritant / corrosive response data:
labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), the test substance should be labelled as: irritating to the skin (R 38).
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on these results and according to the EEC criteria For classification and labelling requirements For dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), 0-129 should be labelled as: irritating to the skin (R 38).
Executive summary:

The study was carried out in accordance with the OECD guideline No. 404, 'Acute Dermal Irritation/Corrosion' and the EEC Directive 92/69/EEC, B.4, 'Acute Toxicity - Skin irritation. Three rabbits were exposed to 0.5 m1 of the test substance, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Observations were made 1, 24, 48 and 72 hours, 7 and 14 days after exposure. Exposure to the test substance resulted in well defined or moderate to severe erythema and slight oedema in the treated skin-areas of the three rabbits. Scaliness was observed in all animals 7 days after exposure. The skin irritation had resolved within 14 days after exposure in all animals. Sticky remnants of the test substance, which became dry remnants after day 1, were present in the treated skin-areas of all animals up to 72 hours after exposure. Dermal application of the test substance resulted in a primary irritation index of 3.7 (moderately irritating) when applied to the intact rabbit skin. Based on these results and according to the EEC criteria For classification and labelling requirements For dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), the test substance should be labelled as: irritating to the skin (R 38).

The test substance would be classified as a category 2 irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classifica tion, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Three New Zealand White rabbits, supplied by David Percival Ltd, Moston,
Sandbach, Cheshire, UK, were used. At the start of the study the animals
weighed 2.64 to 2.97 kg and were twelve to sixteen weeks old. After a
minimum acclimatisation period of five days each animal was given a number
unique within the study which was written with a black indelible marker-pen
on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended metal cages. Free access
to mains drinking water and food (STANRAB SQC Rabbit Diet, Special Diets
Services Ltd, Witham, Essex, UK) was allowed throughout the study.

The animal room was maintained at a temperature of 17 to 20' C and relative
humidity of 46 to 65%. The rate of air exchange was approximately fifteen
changes per hour and the lighting was controlled by a time switch to give
twelve hours continuous light and twelve hours darkness.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
single application into the conjunctival sac of one eye
Observation period (in vivo):
1, 24, 48 and 72 h
Number of animals or in vitro replicates:
3
Details on study design:
Immediately before the start of the test, both eyes of the provisionally selected
test rabbits were examined for evidence of ocular irritation or defect with the
aid of a light source from a standard ophthalmoscope. Animals shOWing
evidence of ocular lesions were rejected and replaced.
One rabbit was initially treated. A volume of 0.1 ml of the test material was
instilled 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 instillation, to prevent loss of
the test material, and then released. The left eye remained untreated and was
used for control purposes. Immediately after administration of the test material,
an assessment of the initial pain reaction was made.
After consideration of the ocular responses produced in the first treated animal,
two additional animals were treated. In order to minimise pain on instillation
of the test material, one drop of local anaesthetic ("Ophthaine", 0.5%
proxymetacaine hydrochloride, E R Squibb & Sons Limited, Hounslow,
Middlesex, UK) was instilled into both eyes of these animals 1 to 2 minutes
before treatment.

Assessment of ocular damage/irritation was made approximately 1 hour and 24,
48 and 72 hours following treatment, according to the numerical evaluation
given in Appendix I, (from Draize J H (1977) "Dermal and Eye Toxicity Tests"
In: Principles and Procedures for Evaluating the Toxicity of Household
Substances, National Academy of Sciences, Washington DC p.48 to 49).
Any other ocular effects were also noted. Examination of the eye was
facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable as no corneal effects were seen
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
2
Reversibility:
other: see above
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0.22
Max. score:
3
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable as no chemosis was seen
Irritant / corrosive response data:
Reversibility of any observed effect: Changes fully reversible within 2 days
Other effects:
None

Summary of ocular lesions

 

Anim.

No.

Effect

Hours

Days after application

Mean

score cornea

Days

1/2/3

Mean score iritis

Days 1/2/3

 

Mean score redness Days 1/2/3

Mean score chemosis Days 1/2/3

1

1

2

3

7

21

129

Cornea*

Iris

Redness

Chemosis

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

0

 

0

 

 

0

 

 

 

0

14

Cornea*

Iris

Redness

Chemosis

0

0

2

1

0

0

1

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

0

 

0

 

 

0.33

 

 

 

0

20

Cornea*

Iris

Redness

Chemosis

0

0

2

1

0

0

1

0

0

0

0

0

0

0

0

0

-

-

-

-

-

-

-

-

0

 

0

 

 

0.33

 

 

 

0

Mean all anim.

 

 

 

 

 

 

 

 

0

 

0

 

0.22

 

0

Interpretation of results:
not irritating
Remarks:
Migrated information not classified Criteria used for interpretation of results: EU
Conclusions:
Because very slight eye irritation was seen which fully disappeared within 2 days, no classification is needed.

The test substance would not be classified as an eye irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

Executive summary:

The study was performed to assess the irritancy potential of the test material following a single instillation to the rabbit eye (Safepharm Standard Method Number 560.01). The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 February 1987) and Method 85 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC). The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 93/21/EEC) relating to the classification, packaging and labelling of dangerous substances.

No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation in two treated eyes one hour after treatment. Minimal conjunctival redness was noted in two treated eyes at the 24-hour observation. Treated eyes appeared normal at the 24 or 48-hour observations.

The test substance would not be classified as an eye irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 10th December 2014 Experimental completion date: 12the December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to
Guideline:
other: OECD Guideline 437 "Bovine corneal Opacity and Permeability Assay"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
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, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.
Vehicle:
unchanged (no vehicle)
Controls:
other: The negative control item, 0.9% w/v sodium chloride solution, was used as supplied. The positive control item, Ethanol, was used as supplied.
Amount / concentration applied:
Volume: 0.75 mL of test item

Concentration: not stated in study report
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
Immediately after exposure
Number of animals or in vitro replicates:
Three corneas to each of the following: test material group, negative control group, positive control group
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 (epithelial side uppermost) 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 (MEM) 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 MEM.

A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer (Appendix 1). 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 MEM 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 MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the 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:
cornea opacity score
Run / experiment:
3 corneas
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: reversibility not determined
Other effects / acceptance of results:
The corneas treated with the test item were clear post treatment and clear 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.

 In VitroIrritancy Score

TheIn Vitroirritancy scores are summarized as follows:

 

Treatment

In VitroIrritancy Score

Test Item

0.1

Negative Control

0.7

Positive Control

52.4

 

 

Criteria for an Acceptable Test

TheIn VitroIrritancy Score of the positive control group narrowly exceeded the upper acceptable range. The upper acceptable range in the Study Plan was 51.0. In this study the positive control result was 52.4. As the positive control value was greater than the acceptance criterion, demonstrating the sensitivity of the test system, and the test itemIn VitroIrritancy Score was clearly ≤3, it was considered that the results of the test are acceptable and a repeat test would be unnecessary.

 

The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The test item, PP-R-001 (CAS#1613243-54-1), was considered to be no category according to the prediction model.
 
Therefore the test item does not require classification to UN GHS or EU CLP.
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 corneain 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, PP-R-001 (CAS#1613243-54-1), 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 anIn VitroIrritancy Score (IVIS).

 

Interpretation

The test item is classified according to the prediction model below:

 

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

TheIn Vitroirritancy scores are summarized as follows:

 

Treatment

In VitroIrritancy Score

Test Item

0.1

Negative Control

0.7

Positive Control

52.4

 

 Conclusion

The test item, PP-R-001 (CAS#1613243-54-1), was considered to be no category according to the prediction model.

 

Therefore the test item does not require classification to UN GHS or EU CLP.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 21 January 2016. Experimental completion date: 18 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study
Qualifier:
according to
Guideline:
other: • OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July, 2015).
Deviations:
no
Qualifier:
according to
Guideline:
other: • MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 14 July 2014.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Human cornea model
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Cell Culture
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA 01721, USA). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).

EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-¬incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 19 hours).
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: De-ionised water Positive control: Methyl acetate
Amount / concentration applied:
the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues.
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
Immediatelky after exposure
Number of animals or in vitro replicates:
Please see below section "Details on Study Design"
Details on study design:
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 rinsing the 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-labeled 12-well plate for 12 minutes 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-labeled 6-well plate containing 1 ml of warm Assay Medium. The tissues are incubated for about 120 minutes at standard culture conditions (post-treatment incubation).

MTT Assay
After post-treatment incubation of about 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 approximately 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.

Each insert was removed from the 24-well plate after approximately 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 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. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for about 2.5 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-labeled 96-well plate.
Remarks on result:
no indication of irritation
Remarks:
Since the viability value of the test item exposed tissues did not decrease below 60% (97.2%), the test item is not considered to possess an eye irritating potential.
Other effects / acceptance of results:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 97.2% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to eye.

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

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.

Results after treatment for 30 minutes withPP-R-001 (CAS#1613243-54-1)and the controls

Dose Group

Mean Absorbance* Tissue 1 and 2 minus Mean Blank

Mean Absorbance of
2 Tissues*

Rel. Absorbance [%]
Tissue 1 and 2*

Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Rel. Absorbance

[% of Negative Control]**

Negative Control

1.989

2.021

98.5

3.1

100.0

2.052

101.5

Positive Control

0.849

0.816

42.0

3.2

40.4

0.784

38.8

Test Item

1.966

1.963

97.3

0.3

97.2

1.960

97.0

Concerning acceptance criteria:

·        The negative control OD is > 1.0 and < 2.6(1.989 and 2.052).

·        The mean relative viability of the positive control is below 50% of the negative control viability (40.4%).

·        The difference of viability between the two relating tissues of a single item is < 20% (values between 0.3% to 3.2%) in the same run (for positive and negative control tissues and tissues of single test items).

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, PP-R-001 (CAS#1613243-54-1) does not possess any eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of PP-R-001 (CAS#1613243-54-1) by means of the Human Cornea Model Test.The OECD test guideline No. 492 was followed.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues 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 > 1.0 and < 2.6 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 60% 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 of a single test item was < 20% in the same run (for positive and negative control tissues and tissues of single test items).

Irritating effects were not observed following incubation with PP-R-001 (CAS#1613243-54-1). Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (97.2%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, PP-R-001 (CAS#1613243-54-1)does not possess any eye irritating potential

Endpoint:
eye irritation: in vivo
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Apparently well conducted GLP study. No certification of analysis included in the report.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:

Albino rabbit, New Zealand White (SPF-Quality)
Source: Broekman Institute, Someren, The Netherlands.
3 male rabbits
Approx. 7-8 weeks

Conditions
Air-conditioned room with approximately 15 air changes per hour and the
environment controlled with optimal conditions considered as being a temperature
of 21°C and a relative humidity of 50%. Fluctuations from these optimal
conditions were noted, but were considered not to have affected study integrity.
Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Accommodation
Individually in labelled cages with perforated floors (Scanbur Denmark) and
eqUipped with an automatic drinking system (ITL, Bergen, The Netherlands).
Acclimatisation period was at least 5 days before start of treatment under
laboratory conditions.

Diet
Standard laboratory rabbit diet (LKK-20, pellet diameter 4 mm, Hope Farms,
Woerden, The Netherlands) approx. 100 g per day. Certificates of analysis were
examined and retained in the NDTDX archives.
In addition, hay (BMI, Helmond, the Netherlands) was provided once a week.

Water
Free access to tap-water diluted with decalcified water.
Certificates of quarterly analysis were examined and retained in the NDTDX
archives.
Controls:
other: other eye served as control
Amount / concentration applied:
The test substance was instilled undiluted as delivered by the sponsor.
Duration of treatment / exposure:
one time instillation
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
A health inspection was performed prior to commencement of treatment, to ensure
that the animals were in a good state of health. Special attention was paid to
the eyes, which were free from any abnormality.

On test day 1, 0.1 ml of the test substance was instilled in the conjunctival
sac of one eye of an animal after gently pulling the lower lid away from the
eyeball. The lids were then gently held together for about one second to prevent
loss of the test substance. The other eye remained untreated and served as the
reference control.

Immediately after the 24 hour observation, a solution of 2% fluorescein in water
(adjusted to pH 7.0) was instilled into both eyes of the animal to
quantitatively determine corneal epithelial damage. Any bright green stained
area, indicating epithelial damage, was estimated as a percentage of the total
corneal area.

The two other animals were treated in a similar manner 7 days later, after
considering the degree of eye irritation observed in the first animal.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: mean score from 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: mean score from 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: mean score fromt 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean score from 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: mean score from 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: score from 24, 48 and 72 hours.
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean from 24, 48 and 72 hours.
Score:
0.33
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: mean from 24, 48 and 72 hours.
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: mean from 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: mean from 24, 48 and 72 hours.
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: mean from 24, 48 and 72 hours
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: mean from 24, 48 and 72 hours.
Score:
0
Max. score:
0
Irritant / corrosive response data:
Instillation of 0.1 ml of the test substance into one eye of each of three rabbits resulted
in irritation of the conjunctivae, which consisted of redness and chemosis. The
irritation had completely resolved within 24 hours in two animals and within 48
hours in the third animal.
No iridic irritation or corneal opacity were observed, and treatment of the eyes
with 2% fluorescein, 24 hours after test sUbstance instillation revealed no
corneal epithelial damage in any of the animals.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on these results and according to the EEC criteria for classification and
labelling requirements for dangerous substances and preparations (Guidelines in
Commission Directive 93/21/EEC, 27th April 1993), the test substance does not have to be
classified and has no obligatory labelling requirement.

The test substance would not be classified as an eye irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.
Executive summary:

The study was carried out in accordance with the OECD guideline No. 405, 'Acute Eye Irritation/Corrosion' and the EEC Directive 92/69/EEC, 8.5, 'Acute Toxicity - Eye irritation. Single samples of 0.1 ml of the test substance were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation. Instillation of 0.1 ml of the test substance into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness and chemosis. The irritation had completely resolved within 24 hours in two animals and within 48 hours in the third animal. Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), the test substance does not have to be classified and has no obligatory labelling requirement.

The test substance would not be classified as an eye irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN:

The corrosivity potential of the test item, CAS#1613243 -54 -1, was evaluated 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 were used to make a prediction of the corrosivity potential of the test item. It was concluded that CAS#1613243 -54 -1 was considered to be non-corrosive to the skin. 

The second in vitro test evaluated the skin irritation potential of the test item, CAS#1613243 -54 -1, using the EPISKINTM reconstructed human epidermis model (a treatment period of 15 minutes followed by a postexposure incubation period of 42 hours). 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 MTT tetrazolium salt in the test item treated tissues relative to the negative controls. The relative mean viability of the test item treated tissues was 54.1% after the 15Minute exposure period and 42hours postexposure incubation period. The test item was classified as non-irritant. The following classification criteria apply: EU DSD and CLP: Not classified for Irritation; UN GHS: Not classified for Irritation (category

Two invivo studies on CAS# 24748 -23 -0 (one of the major constituents of CAS# 1613243 -54 -1, which is being submitted for registration) are available. In a key skin irritation study, that test material produced positive criteria in 3/3 rabbits according to the EU labelling regulations, Annex VI of Council Directive 67/548/EEC, Relating to the Classification, Packaging and Labelling of Dangerous Substances, and was classified as irritant to rabbit skin. However, based on a mean erythema score of 2 and a mean edema score of 1 and full reversibility of the effects within 14 days, the test substance did not meet the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012. In the second study, the test material (CAS# 24748-23-0) exposure resulted in well-defined or moderate to severe erythema and slight edema in the treated skin-areas of the three rabbits. Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), this test material should be labelled as: irritating to the skin. The test material would be classified as a category 2 irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

Conclusion: Although the two supporting studies suggested a mild skin irritating property of one of the constituents of the current test substance (CAS# 1613243 -54 -1), direct in vitro skin irritation testing of the latter substance showed it to be a non-corrosive (OECD 431) as well as non-irritant (OECD 439) to the skin. It is possible that formulation differences (lower concentration of CAS# 24748 -23 -0 in CAS# 1613243 -54 -1) may explain the lack of skin irritating property of CAS# 1613243 -54 -1.

EYE:

Two invtro tests on CAS#1613243 -54 -1 concluded that the test substance is not a corrosive or not an eye irritant. 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. Damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The method can correctly identify test items inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Items and EU Classification, Labelling and Packaging of chemicals (Regulation (EC) No 1272/2008). Test 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. The undiluted test item (CAS#1613243 -54 -1) 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. Based on the results, the test item was considered to be no category according to the prediction mode i.e., test item (CAS#1613243 -54 -1) does not require classification to UN GHS or EU CLP.

An in vitrostudy was performed to assess the eye irritation potential of PP-R-001 (CAS#1613243-54-1) by means of the Human Cornea Model Test. (OECD test guideline No. 492). 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. Treatment with the positive control induced a decrease below 60% compared with the negative control value in the relative absorbance. Irritating effects were not observed following incubation with CAS#1613243 -54 -1. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (97.2%). It was concluded that under the experimental conditions reported, CAS#1613243-54-1does not possess any eye irritating potential.

Additionally, two invivo studies on CAS# 24748 -23 -0, one of the major constituents of CAS# 1613243 -54 -1, conluded that the constituent would not be classified as an eye irritant. In one eye irritation study, no corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation in two treated eyes one hour after treatment. Minimal conjunctival redness was noted in two treated eyes at the 24 -hour observation. Treated eyes appeared normal at the 24 or 48-hour observations. The test substance would not be classified as an eye irritant in accordance with the criteria for classification in accordance with the Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 Nov, 2012. In the second eye irritation study, instillation of 0.1 ml of the test substance into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness and chemosis. The irritation had completely resolved within 24 hours in two animals and within 48 hours in the third animal. Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC, 27th April 1993), the test substance does not have to be classified and has no obligatory labelling requirement. This test substance would not be classified as an eye irritant in accordance with the criteria for classification in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 3.0 November 2012.

Conclusion: Thus, two invitro studies for eye irritation on CAS# 1613243 -54 -1 (BCOP test (OECD ) and the EpiOcular test (OECD 492)) concluded the test substance to be non-irrtanrt to the eye. Additionally, two invivo eye irritation studies on CAS# 24748 -23 -0 (one of the major constituents of the current test item)concluded that classification as an eye irritant would not be required.

Justification for selection of skin irritation / corrosion endpoint:

A well conducted in vivo GLP study on one of the major constituents of the test item.  

Justification for selection of eye irritation endpoint:

.A well conducted in vivo GLP study on one of the major constituents of the test item.

Justification for classification or non-classification

The test substance, 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives, did not meet the criteria for classification as a skin irritant and did not meet the criteria for classification as an eye irritant as described above. These data are conclusive, and no classification for the test item is needed on skin or eye irritation.