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Administrative data

Description of key information

- Episkin test for skin irritation (OECD 439): 83% cell viability: Non irritant

- BCOP (Bovine Corneal Opacity and Permeability) test (OECD 437): IVIS 18.5, No prediction can be made

- EpiocularTM(OECD 492): 77 % cell viability, Non irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-25 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
Version 1.8 (February 2009)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiSkin Model has been validated for irritation testing in an international trial. The EpiSkin method is a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Remarks:
Epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 16-EKIN-047
- Expiry date: 28 November 2016
- Date of initiation of testing: 23 November 2016

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, >= 95% humidified atmosphere.

EXPOSURE
- Test Item: The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
- Positive and negative control: A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Triplicates

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1x solution, once. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source.
- Observable damage in the tissue due to washing: none

POST-INCUBATION
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, >=95% humidified atmosphere.

MTT TEST
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch(supplied as part of the kit), The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (0.04N HCl), mixed by using a vortex mixer and incubated for 4 hours at room temperature protected from light with gentle agitation (~150 rpm). At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIAVILITY MEASUREMENTS
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA:
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: 1x PBS (Phosphate Buffered Saline)

POSITIVE CONTROL
- Amount(s) applied:10 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5% aq. solution
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
Three replicates were used for the test item and controls, respectively.
Irritation / corrosion parameter:
% tissue viability
Value:
83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean viability value of three tissues
Other effects / acceptance of results:
OTHER EFFECTS:
- Possible direct MTT reduction with test item
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded. :
- Colouring potential of test item
The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is colourless and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD value 0.967 and standard deviation value (SD) for the % viability 7.42 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 10% mean viability range standard deviation value (SD) for the % viability 0.76 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 9.65 % SD

Cell viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

Negative Control (1xPBS):

 Replicate  Optical Density (OD)  Viability (%)
 1  0.920  95
 2 0.931 96
 3  1.049 109
 Mean  0.967  100

Standard Deviation (SD)

   7.42

Positive Control (SDS 5% aq.):

 Replicate  Optical Density (OD)  Viability (%)
 1  0.098  10
 2  0.086  9
 3  0.100  10
 Mean

 0.094

 10
 Standard Deviation (SD)    0.76

Test item:

 Replicate  Optical Density (OD)  Viability (%)
 1  0.694  72
 2  0.865  89
 3  0.844  87
 Mean  0.801  83
 Standard Deviation (SD)    9.65
Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item ATP, Di-Na (CAS 987-65-5) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).
Executive summary:

EpiSkinTM SM test of ATP, Di-Na (CAS 987-65-5) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item ATP, Di-Na did not show significantly reduced cell viability in comparison to the negative control (mean value: 83 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. Positive and negative control values were within the corresponding historical control data ranges.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. The test item ATP, Di-Na (CAS 987-65-5) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
weight of evidence
Study period:
29-30 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: ABP, Perth, PH1 3XB
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to test facility was on the same day as slaughter. Cold packs were used to keep the contents cool.
- Time interval prior to initiating testing: Corneas were prepared and used for testing on the day of collection
- indication of any existing defects or lesions in ocular tissue samples: Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use.Any eyes showing defects were rejected from further use.
- Indication of any antibiotics used: Not specified
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration Test Item: 20.06%, w/v in physiological saline
NEGATIVE CONTROL: Physiological saline
- Amount(s) applied: 750 µL
- Concentration: Sodium Chloride 0.9%, w/v
- Batch no.: 16B29T2C
POSITIVE CONTROL : Imidazole
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20.03%, w/v in physiological saline
- Batch no.: SLBP2962V
Duration of treatment / exposure:
4 h ± 10 min
Number of animals or in vitro replicates:
Three Corneas per test item and control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of ca 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use. Prior to mounting corneas the width of the cornea was measured using a ruler.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were then mounted, epithelial side forward, into pre-warmed corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed EMEM (Eagles Minimum Essential Medium) without phenol red. The posterior chamber was filled first to encourage the cornea to return to its original curvature, and care was taken to avoid the introduction of bubbles into the medium. Holders were then allowed to equilibrate for >1 h in an incubator.
Unless otherwise stated, all incubations of corneas were conducted in an incubator set to maintain 32°C (actual temperatures have been recorded in the raw data). At the end of the equilibration period, the medium in both chambers was replaced with fresh, EMEM without phenol red. Baseline opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Damaged corneas (opacity >7 opacity units) were rejected from further use.

TREATMENT METHOD: closed chamber. Prior to dosing, cornea holders were tipped forward to avoid contact between the dosing solution and the corneal epithelium. ATP, Di-Na (20.06%, w/v in physiological saline; 750 µL) was applied to the surface of three corneas using syringe. The negative control, physiological saline (750 µL) and the positive control, imidazole (20.03%, w/v in physiological saline; 750 µL), were also applied to the surface of three corneas each, using a syringe. The anterior chambers were then sealed with tape. To begin exposure, the corneal holders were tilted to a horizontal position, taking care to ensure that the entire epithelial surface was covered with the dosing solution. The dosed units were then returned to the incubator for 4 h ± 10 min.

REMOVAL OF TEST SUBSTANCE
EMEM with and without phenol red were pre-warmed prior to use. Following the exposure incubation, the test items and control solutions were removed from the anterior chambers through the holes using a vacuum pump. Corneas were then rinsed three times with EMEM with phenol red (ca 5 mL per rinse), removing the EMEM each time, then rinsed once with EMEM without phenol red (ca 5 mL), which was also removed. Finally, both chambers were refilled with EMEM without phenol red, and any bubbles introduced into the medium were removed.

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Gross damage or other changes were observed by visual assessment.
- Corneal permeability: Following the measurement of opacity, the EMEM in both chambers of the cornea holder wasremoved. Posterior chambers were refilled with EMEM without phenol red and sealed. Sodium fluorescein solution (5 mg/mL in EMEM without phenol red; ca 1 mL) was added to the anterior chambers. Cornea holders were rotated to the horizontal position, and incubated for 90 min ± 5 min in an incubator set to maintain a temperature of 32°C. At the end of the permeability test, EMEM from the posterior chambers was collected. These samples were stored overnight in a refrigerator set to maintain a temperature of 4°C.
Following overnight storage, triplicate aliquots from each sample (380 µL) were transferred into a 96 well plate, and analysed with a Multiskan Spectrum plate reader at 490 nm. Three blank wells containing EMEM without phenol red (380 µL) were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluorescein, 10 µg/mL).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = Opacity change + (15 x A490 Value)

DECISION CRITERIA:
In accordance with OECD Test Guideline No. 437, irritancy was assigned on the basis of in vitro irritancy scores (IVIS), calculated from the opacity and permeability value for each cornea, as follows:

IVIS<=3: not classified according to GHS/CLP
3IVIS>55: Classified as Eye Damaging Category 1 according to GHS/CLP
Irritation parameter:
in vitro irritation score
Run / experiment:
Single experiment
Value:
18.5
Vehicle controls validity:
valid
Remarks:
IVIS: 0.0
Positive controls validity:
valid
Remarks:
IVIS: 106.60
Remarks on result:
not determinable
Remarks:
No Prediction can be made
Other effects / acceptance of results:
OTHER EFFECTS:
- Test item treatement: Prior to baseline opacity checks cornea diameter was measured and the mean diameter for the 3 test item treated corneas was 2.23 cm.
The BCOP results were similar for all corneas and are presented in full in Table 1 (see Any other information on results incl. tables section).
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Negative and Positive Control Treatments: Prior to baseline opacity checks cornea diameter was measured. Negative and positive control corneas had a mean diameter of 2.33 cm and 2.23 cm, respectively. The negative control treatment gave opacity readings <5 and low permeability values. The classification achieved was “No Category”. The mean change in opacity after treatment was 1.53. The mean permeability (corrected for blank only) was 0.072. The positive control treatments gave mean IVIS scores of 106.60. The classification achieved was “Category 1”.
- Acceptance criteria met for negative control: yes, mean post treatment opacity of 1.53 (historical values range 3.83 +/- 1.82) / mean permeability corrected for blank of 0.072 (historical values range 0.045 +/- 0.043)
- Acceptance criteria met for positive control: yes, mean IVIS 106.60 (historical range: Mean +/- 2SD =66.29 to 169.36)

Table 1                         BCOP Results for Controls and ATP, Di-Na (n=3)

Treatment

Opacity Post Dose

(Opacity Units, Uncorrected)

Corrected* Opacity Change

(Opacity Units)

Permeability (A490), Corrected for Blank Only

Permeability (A490), Corrected*

IVIS

IVIS (Mean)

UN GHS Category**

Physiological Saline (Negative Control)

1.26

-1.96

0.020

-0.053

-2.75

0.00

No Category

4.72

2.00

0.162

0.090

3.35

3.98

-0.05

0.036

-0.037

-0.60

Imidazole (PositiveControl)

93.23

90.17

1.672

1.600

114.16

106.60

Category 1

87.47

84.61

1.499

1.427

106.01

80.93

78.42

1.486

1.414

99.63

ATP, Di-Na

17.52

15.02

0.010

-0.063

14.08

18.50

No Prediction can be made

22.18

19.04

0.008

-0.065

18.07

27.67

24.31

0.007

-0.066

23.33

* Corrected for Negative Control

**Classifications from OECD Guidelines for the Testing of Chemicals No. 437 (2013)

Interpretation of results:
other: no prediction could be made
Conclusions:
In conclusion, no prediction could be made for ATP, Di-Na (CAS 987-65-5) using the BCOP assay according to the UN GHS classifications and the CLP classification system.
Executive summary:

The objective of this study was to evaluate the ocular irritation potential of ATP, Di-Na (CAS 987-65-5) using the Bovine Corneal Opacity and Permeability (BCOP) assay (OECD guideline 437).

Corneas were dissected from freshly obtained cow eyes, the cornea diameter was measured and they were mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated as follows:

ATP, Di-Na (20%, w/v; 750 µL), physiological saline or imidazole (20%, w/v) as negative and positive controls, respectively, were applied to three corneas each. Following exposure for 4 h ± 10 min, the test or control items were rinsed off.

The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The results are summarised as follows:

The mean IVIS Score for ATP, Di-Na was 18.50

In conclusion, no prediction could be made for ATP, Di-Na (CAS 987-65-5) using the BCOP assay according to the UN GHS classifications and the CLP classification system.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04-05 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; for the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model.
Version / remarks:
10 Feb. 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: The EpiOcular™ human cell construct (MatTek Corporation)
Details on test animals or tissues and environmental conditions:
DETAILS ON TISSUE MODEL:
EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
-Lot No.:27007
- Expiry date:05 October 2017
- Storage: The EpiOcular™ (OCL-200-EIT) units were stored at refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
Amount applied : 50 mg
Duration of treatment / exposure:
6 h (± 15 min)
Duration of post- treatment incubation (in vitro):
25 ± 2 minutes immersion incubation (Post-Soak)
18 h ± 15 minutes (Post-treatment Incubation)
Number of animals or in vitro replicates:
Two
Details on study design:
DETAILS ON THE TEST PROCEDURE USED:

- Preparation of EpiOcular™ Tissues for Treatment
After the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes. The Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37°C in an incubator with 5±1% CO2, 90±10% humidified atmosphere 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 (16 - 24 hours).

- Application
Two replicates were used for the test item and control(s) respectively.
Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Test Item: Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface (e.g. the lid of a microtiter plate) and dosed by pouring the solid test article onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
Positive and Negative Control: A volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface if necessary.

- Exposure
The plates with the treated tissue units were incubated for the exposure time of 6 hours
(± 15 min) at standard culture conditions (37±1°C in an incubator with 5±1% CO2, 90±10%
humidified atmosphere).

- Rinsing
After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as following way: Three clean beakers (glass or plastic with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS were used per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care of not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was 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 tissue was 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. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

-Post-Soak and Post-incubation
After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 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 insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18h ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, 90±10% humidified atmosphere.

- Formazan Extraction
Each insert was removed from the 24-well plate after 3 hours ± 10 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with parafilm (between the plate cover and upper edge of the wells)
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were
treated identically.


- Cell Viability Measurements
Following the formazan extraction, 200 µL sample(s) from each tube (2×200 µL) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using isopropanol solutions as the blank (8×200 µL).


OTHER INFORMATION ON MATERIALS AND METHODS

- Check-method for Possible Direct MTT Reduction by the Test Item
Approximately 50 µL test item was added to 1 mL MTT 1 mg/mL solution in a tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere. A negative control (50 µL of sterile deionized water) run concurrently. The mixture was incubated for approximately three hours and then any colour change observed:
- Test item which do not interact with MTT: yellow
- Test item interacting with MTT: blue or purple
If the MTT solution’s colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed EpiOcular tissues).

- Check-method to Detect the Colouring Potential of Test Item
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. Blue, dark purple and black test articles may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue MTT product.
Approximately 50 µL test item was added to 1mL of water in a tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere for one hour and then colour checked (unaided eye assessment).
Approximately 50 µL test item was added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in tubes. The tube was placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature and then colour checked (unaided eye assessment).


- Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. OD reading was made following the same conditions as for the other tissues.



- Assay acceptance criteria
- The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is:
30 minute exposure: below 50% of control viability (liquids)
6 hours exposure: below 50% of control viability (solids)
- The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: Mean Tissue Viability (% of negative control)
Value:
77
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item (see section 5.5.1). The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.

Colouring potential of test item:
The test item showed no ability to become coloured in contact with water. However, the isopropanol became opalescent after the contact with test item, two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.022. The Non Specific Colour % (NSC %) was calculated as 1.66 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD value 1.344
- Acceptance criteria met for positive control: 38 % viability at 6 hours exposure
- Difference of viability between the two tissue replicates: 0.8 %- 9.3%.

Cell Viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

OD values and viability percentages of the controls:

Negative control: Sterile deionized water

 Replicate  Optical Density (OD)  Viability (%)

 Delta %

 1

1.393 

 104  7.3
 2  1.295 96   7.3
 mean  1.344  100

 

Positive control: Methyl acetate

 Replicate  Optical Density (OD)  Viability (%)  Delta %
 1 0.469  35  6.2
 2  0.552  41  6.2
 mean  0.510  38  

OD values and viability percentages of the test item: ATP, Di-Na

 Replicate  Optical Density (OD)  Viability (%)  Delta %
 1  1.091 81  9.3
 2  0.966  72  9.3
 mean  1.092  77  
 Standard Deviation (SD)    6.59  

OD values and NSC % of additional control: ATP, Di-Na

 Additional colour control Optical Density (OD)  Non Specific Colour % (NSC living%)  delta%
 1  0.028  1.66  0.8
 2  0.017  1.66  0.8
 mean  0.022  1.66  

Remark: Delta %: The difference of viability between the two relating tissues

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, ATP, Di-Na is (CAS No. 987-65-5), thus, considered as non-irritant to eye (UN GHS No Category).

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test itemATP, Di-Na (CAS No. 987-65-5)on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before treatment the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37± 1°C in an incubator with 5± 1% CO2, 90±10% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37± 1°C in an incubator with 5± 1% CO2protected from light, 90±10% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item showed no ability to become coloured in contact with water. However, the isopropanol became opalescent after the contact with test item. Based on this information additional controls and data calculation were necessary.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (=) to 60% compared to the negative control. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

The test item ATP, Di-Na did not show significantly reduced cell viability in comparison to the negative control (mean viability: 77 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

 

The results obtained from thisin vitroeye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, ATP, Di-Na(CAS No. 987-65-5)is, thus, considered as non-irritant to eye (UN GHS No Category).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

A valid EpiSkinTM SM test of ATP, Di-Na was performed under GLP to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

EPISKIN discs were exposed in triplicate to ATP, Di-Na for 15 min at room temperature. The EPISKIN discs treated with the test item did not show significantly reduced cell viability (83% viability compared to the negative controls). Therefore ATP, Di-Na is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category / CLP not classified). The study is rated as key study and as Klimisch 1 "reliable without restriction".

Eye irritation:

A valid assessment of ocular irritation of ATP, Di-Na In Vitro Using the Bovine Corneal Opacity and Permeability Assay was performed under GLP according the the OECD guideline Nr, 437, 26 July 2013.

ATP, Di-Na ( 20%, w/v in physiological saline; 750 µL), was applied to three corneas. Following exposure for 4 h ± 10 min, it was rinsed off. The in vitro irritancy score (IVIS) calculated for this substance was 18.5. If the IVIS score is </= 3, the substance does not require classification for eye damage according to the UN GHS classifications and the CLP classification system. If the IVIS score is >/= 55, the substance has to be classified as corrosive to the eye (Category 1). If the IVIS score is between these two values no prediction can be made and further testing is necessary.

In conclusion, the BCOP assay categorised ATP, Di-Na as ‘No prediction can be made’ according to the UN GHS classifications and the CLP classification system. The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

A valid study was performed under GLP to determine the acute ocular irritation potential of the test item ATP, Di-Na on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to OECD guideline Nr. 492, 28 July 2015.

EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±1°C) in an incubator with 5±1 % CO2, 90±10% humidified atmosphere). The Epiocular disks treated with ATP, Di-Na did not show significantly reduced cell viability, </= 60%, in comparison to the negative control (mean relative viability: 77 %). The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item a ATP, Di-Na indicated that the test item is, thus, considered as non-irritant to eye (UN GHS No Category). The study is used in a weight of evidence approach and is rated as Klimisch 1 "reliable without restriction".

Justification for classification or non-classification

For skin irritation:

According to the results obtained with the EpiSkinTM SM test (OECD 439) ATP, Di-Na (CAS No. 987-65-5) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category /CLP not classified).

For Eye Irritation:

No prediction could be made for ATP, Di-Na (CAS No. 987-65-5) using the BCOP assay (OECD 437) according to the UN GHS classifications and the CLP classification system. Therefore a second test was performed.

Acording to the results obtained with the Epiocular test (OECD 492) a ATP, Di-Na (CAS No. 987-65-5) is considered to be non-irritant to the eyes and is therefore not classified (UN GHS No Category / CLP not classified).