Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: not corrosive and not irritating in vitro (OECD 431 and 439, GLP)
Eye irritation: not irritating (OECD 405, GLP and OECD 437, GLP)
Respiratory irritation: no study available

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 - 29 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ in vitro reconstructed human epidermis model
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: not specified
- Delivery date: 27 July 2010
- Date of initiation of testing: the study was performed between 27 and 29 July 2010

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS with Ca++ an Mg++ to gently remove any residual test material. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 35%, or if the viability after 3 minutes exposure is greater than or equal to 35% and the viability after 1 hour exposure is less than 35%. The test substance is considered to be corrosive to skin if the viability after 60 minutes exposure is greater than or equal to 35% and the viability after 240 minutes exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if viability after 240 minutes exposure is greater than or equal to 35%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 mg
- Other: 100 µl of 0.9% w/v sodium chloride solution was added for wetting of the test item.

VEHICLE
no vehicle used

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 0.9% (w/v) sodium chloride solution

POSITIVE CONTROL
- Amount(s) applied: 50 µL glacial acetic acid
Duration of treatment / exposure:
Test material: 3, 60 and 240 min
Negative and positive controls: 240 min
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
duplicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
117.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
135.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 min
Value:
105.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not specified
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: not specified

Mean OD540 values and viabilities for the negative control, positive control and test material are given in Table 1.
The qualitative evaluation of tissue viability is given in Table 2. Following the 3, 60 and 240 min exposure periods the test material treated tissues appeard blue which was considered to be indicative of viable tissue.

Table1 :Mean OD540Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

Exposure period

Mean OD540of duplicate tissues

Relative mean viability (%)

Negative control**

240 minutes

0.161

100*

Positive control**

240 minutes

0.005

3.1

Test substance

240 minutes

0.170

105.6

60 minutes

0.218

135.4

3 minutes

0.189

117.4

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

** = Control group shared with Harlan Laboratories Ltd Project numbers 2920/0150, 2920/0151, 2920/0153, 2920/0154 and 2920/0155

Table2 : Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

 

Item

Exposure period

Mean OD540of duplicate tissues

Relative mean viability (%)

Negative control*

240 minutes

-

-

Positive control*

240 minutes

++

++

Test substance

240 minutes

-

-

60 minutes

-

-

3 minutes

-

-

- = Blue tissue (viable)

+ = Blue/white tissue (semi-viable)

++ = Tissue completely white (dead)

* = Control group shared with Harlan Laboratories Ltd Project numbers 2920/0150, 2920/0151, 2920/0153, 2920/0154 and 2920/0155

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 3.1% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean cell viability of the test material treated tissues was 117.4, 135.4 and 105.5% after 3, 60 and 240 min exposure, respectively. Under the conditions of this study, the test material is thus considered to be not corrosive in vitro. Therefore, the test material does not meet the criteria for classification for Skin corrosion Category 1 according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS).

CLP: not classified
GHS: not classified
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 12 Nov 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ reconstructed human epidermis model
- Tissue batch number(s): 12-EKIN-041
- Production date: not specified
- Shipping date: not specified
- Delivery date: 06 Nov. 2012
- Date of initiation of testing: The study was performed between 06 Nov. and 12 Nov 2012.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+. 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.
- Observable damage in the tissue due to washing: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT solution
- Incubation time: 3 hours at 37°C
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: without reference filter

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

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

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant (Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)
Relative mean tissue viability ≥ 50%: Non-irritant (Not classified according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg on 0.38 cm² (corresponds to 26.3 mg/cm²)

VEHICLE
-no vehicle used

NEGATIVE CONTROL
- Amount(s) applied: 10 µL Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+


POSITIVE CONTROL
- Amount(s) applied: 10 µl odium dodecylsulfate
- Concentration: 5% (w/v)
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min
Value:
112.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
5.4% tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viability values and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.The relative mean viability of the test item treated tissues was 112.2% after a 15-minute exposure period.
Other effects:
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 8.2%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.697. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 9.4%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

±SDof OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Material

0.728

0.697

0.057

104.4

100*

8.2

0.631

90.5

0.732

105.0

Positive Control Material

0.032

0.038

0.015

4.6

5.4

2.2

0.055

7.9

0.026

3.7

Test Material

0.855

0.782

0.065

122.7

112.2

9.4

0.729

104.6

0.761

109.2


SD=    Standard deviation

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

⊕ = Control group shared with Harlan Laboratories Ltd, Project numbers 41205083, 41205113, 41205120, 41205125, 41205128 and 41205133


Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the test item treated tissues was 112.2% after a 15-minute exposure period. Under the conditions of this study, the test item is thus considered to be not irritating in vitro. Therefore, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS).

CLP: not classified
GHS: 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 vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 10 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
other: New Zealand White (Hsdlf:NZW)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.61-2.93 kg
- Housing: The animals were individually housed in suspended cages. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet: 2930C Teklad Global Rabbit diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
single instillation without washing
Observation period (in vivo):
72 h
Reading time points: 1, 24, 48 and 72 h
Number of animals or in vitro replicates:
2

Details on study design:
SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: ophthalmoscope
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: reversibility not applicable
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: reversibility not applicable
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritant / corrosive response data:
Individual scores for ocular irritation are given in Table 1. The individual and mean scores as required for EU and GHS classification and labelling are presented in Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in both treated eyes one hour after treatment.
Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye one hour after treatment. Minimal conjunctival irritation noted in both treated eyes at the 24-hour observation.
Both treated eyes appeared normal at the 48-hour observation.
Other effects:
Individual bodyweights and bodyweight changes are given in Table 3.
Both animals showed expected gain in bodyweight during the study.
No further local or systemic effects were observed during the study period.

Table 1. Individual Scores for Ocular Irritation

Rabbit Number and Sex

72824 Male

72839 Male

IPR = 1

IPR =2

Time After Treatment [h]

1

24

48

72

1

24

48

72

CORNEA

Degree of opacity

Area of cornea involved

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

IRIS

1

0

0

0

1

0

0

0

CONJUNCTIVAE

Redness

Chemosis

Discharge

 

1

1

1

 

1

1

0

 

0

0

0

 

0

0

0

 

1

2

2

 

1

1

1

 

0

0

0

 

0

0

0

 

IPR = Initial Pain Reaction

 

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

Rabbit Number and Sex

Time After Treatment [h]

Corneal Opacity

Iridial Inflammation

Conjunctival Redness

Conjunctival Chemosis

72824 Male

24

48

72

0

0

0

0

0

0

1

0

0

1

0

0

Mean

0.0

0.0

0.3

0.3

72839 Male

24

48

72

0

0

0

0

0

0

1

0

0

1

0

0

Mean

0.0

0.0

0.3

0.3

 

Table 3. Individual Body Weights and Body Weight Changes

Rabbit Number and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

Day 0

Day 3

72824 Male

2.93

3.00

0.07

72839 Male

2.61

2.68

0.07

Interpretation of results:
GHS criteria not met
Conclusions:
The test item produced individual scores of 0/0 for corneal opacity, 0/0 for iritis, 0.3/0.3 for conjunctival redness and 0.3/0.3 for chemosis, calculated as the mean scores following grading at 24, 48 and 72 hour after instillation. Observed effects were fully reversible within the observation period.
Therefore, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the Globally Harmonised System of Classification and Labelling of Chemicals.
The test item is thus considered to be not irritating to rabbit eyes.

CLP: not classified
GHS: not classified
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Dec 2012
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”.
The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.
IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Time interval prior to initiating testing: max. 24 h
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) supplemented with penicillin/streptomycin, on ice
PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: 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.
- Test medium and temperature conditions used in the cornea holder: complete minimum essential medium (MEM) at 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects, initial opacity of 1-6 opacity units
DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea using a calibrated opacitometer.
Vehicle:
other: 0.9% w/v sodium chloride solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied in the test: 750 μL
- Concentration (if solution): 20% w/v suspension in 0.9% w/v sodium chloride solution
NEGATIVE CONTROL SUBSTANCE
- Substance: sodium chloride
- Concentration: 0.9% w/v in water
- Amount(s) applied in the test: 750 μL
POSITIVE CONTROL SUBSTANCE
- Substance: imidazole
- Concentration: 20% w/v in 0.9% w/v sodium chloride solution
- Amount(s) applied in the test: 750 μL
Duration of treatment / exposure:
240 min
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
none
Number of animals or in vitro replicates:
number of corneas: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: closed-chamber method
The MEM was removed from the anterior chamber of the BCOP holder and 750 µL of the test item or control items were applied to the cornea. 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°C for 240 min.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test and control substances were removed from the anterior chamber and the epithelium washed four times. The anterior and posterior chambers were refilled with fresh complete MEM.
- Medium for washing the corneas: complete MEM containing phenol red
- Medium for final rinsing: complete MEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea using a calibrated opacitometer.
- Time of determination: Final opacity reading was taken at the end of the 240-minute post-exposure incubation period, following the final rinsing.

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: The medium of the anterior chamber was removed and sodium fluorescein solution was added. The dosing holes were plugged prior to holders' incubation (anterior chamber uppermos). The amount of sodium fluorescein that crossed into the posterior chamber was measured by means of spectrophotometry at 492 nm and recorded as optical density (OD492).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL)
- Incubation time: 90 min at 32 ± 1°C
- Treatment for measuring: OD492 of a 360 µL aliquot was determined in a 96-well plate.
- Dilution of the medium: If values greater than 1.500 OD492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

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

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

- Opacity Measurement: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

- Permeability Measurement: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

- In Vitro Irritancy Score: The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

- Visual Observation: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

DATA INTERPRETATION
A test item that induces an In Vitro Irritancy Score ≥ 55.1 is defined as an ocular corrosive or severe irritant and will be classified for irreversible effects on the eye (Category 1) according to EU CLP (Regulation (EC) No 1272/2008) and UN GHS, and for risk of serious damage to eyes (R41) according to the Dangerous Substance Directive (Council Directive 67/548/EEC).

CRITERION FOR AN ACCEPTABLE TEST
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fel within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 55.8 to 126.1.
0.9% w/v sodium chloride solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the highest In Vitro Irritancy Score for the testing facility during 2012 up to commencement of this study ≤ 6.1.
Irritation parameter:
in vitro irritation score
Run / experiment:
240 min
Value:
10.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.
The condition of each cornea post treatment is given in Table 2.
Two corneas treated with the test item were clear with cloudy areas and one was slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

IN VITRO IRRITANCY SCORE

 

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Test item

10.9

Negative control

4.2

Positive control

105.5

 

CRITERION FOR AN ACCEPTABLE TEST

The positive control In Vitro irritancy Score was within the range of 55.8 to 126.1. The positive control acceptance criterion was therefore satisfied.

The negative control In Vitro irritancy Score was 4.2. The negative control acceptance criterion was therefore satisfied.

 

 

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In vitroIrritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

3

8

5

 

0.032

 

 

2

1

4

3

 

0.047

 

 

3

2

5

3

 

0.031

 

 

 

 

 

3.7*

 

0.037^

 

4.2

Positive Control

4

3

75

72

68.3

2.495

2.458

 

5

4

80

76

72.3

2.260

2.223

 

6

4

70

66

62.3

2.930

2.893

 

 

 

 

 

67.7¤

 

2.525¤

105.5

Test Material

10

4

18

14

10.3

0.012

0.000

 

11

6

25

19

15.3

0.076

0.039

 

12

4

14

10

6.3

0.031

0.000

 

 

 

 

 

10.7¤

 

0.013¤

10.9

 

OD= Optical density

* = Mean of the post treatment-pre‑treatment values

^= Mean permeability             

¤ = Mean corrected value

Table 2. Corneal Epithelium Condition

 

Treatment

Cornea Number

Observation

Post Treatment

Negative Control

1

clear

2

clear

3

clear

Positive Control

4

cloudy

5

cloudy

6

cloudy

Test Material

10

clear with cloudy areas

11

slightly cloudy

12

 clear with cloudy areas

 

Interpretation of results:
study cannot be used for classification
Conclusions:
The mean In Vitro Irritancy Score of the test material treated corneas was 10.9 after a 240 min exposure. Under the conditions of this study, the test material is thus considered to be not corrosive or a severe irritant in vitro. Therefore, the test material does not meet the criteria for classification for Severe Eye Damage Category 1 according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS).
No conclusion can be made whether the substance is an eye irritant or not.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

Skin corrosion (in vitro)

The corrosivity potential of dicalcium pyrophosphate was tested using the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model following OECD Guideline 431 and complying with GLP. Duplicate tissues were treated with 20 mg of the test material for 3, 60 and 240 min. Duplicate tissues treated for 240 min with 50 µL 0.9% w/v sodium chloride or glacial acetic acid served as negative and positive controls, respectively. At the end of the exposure period, tissues were rinsed prior to MTT loading. Formazan crystals were extracted from the MTT loaded tissues by acidic isopropanol extraction. The optical density of the extracts was measured at 540 nm. The relative mean viability (MTT reduction to formazan in treated vs. negative control tissues) was calculated as percent mean optical density of the isopropanol extracts from treated tissues relative to the negative control.

The relative mean viability of tissues treated with the test material was 117.4, 135.4 and 105.6% after 3, 60 and 240 min, respectively. The relative mean viability of the positive control treated tissues was 3.1% after 240 min.

Therefore, based on the study results, the test material does not meet the criteria for classification for Skin corrosion Category 1 according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS), and is thus considered to be not skin corrosive in vitro.

Skin irritation (in vitro)

In another GLP-compliant in vitro study, the skin irritation potential of dicalcium pyrophosphate was evaluated using the EPISKIN™ RHE Model according to OECD Guideline 439 and EU Method B.46. Triplicate tissues were treated with 10 mg of the test substance for 15 min, followed by rinsing and a 42 h post-exposure incubation period. Triplicate tissues concurrently treated with 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ or Sodium Dodecyl Sulphate (SDS) 5% w/v served as negative and positive controls, respectively. Following the post-exposure period, MTT tissue loading and determination of relative mean viability was performed as described above under ‘Skin corrosion (in vitro)’.

The relative mean viability of the test substance-treated tissues was 112.2% of the negative control value, while the relative mean tissue viability of the positive control was 5.4%.

Therefore, based on the study results, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS), and is thus considered to be not skin irritating in vitro.

In support of this notion, no skin irritation was observed in guinea pigs topically treated with dicalcium pyrophosphate in a skin sensitisation study. This suggests that the substance is likely to be not skin irritating in vivo.

Based on the negative results of the above results from valid in vitro skin corrosion and irritation studies, along with supporting evidence from an in vivo skin sensitisation study, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not skin irritating.

Eye irritation/corrosion

A Bovine Corneal Opacity and Permeability (BCOP) Assay was conducted with dicalcium pyrophosphate following OECD Guideline 437 and in accordance with GLP. Three corneas were treated with the test substance at 20% w/v in 0.9% w/v sodium chloride solution for 240 min at 32°C. Two groups of three corneas each treated with 0.9% w/v sodium chloride and 20% w/v imidazole in 0.9% w/v sodium chloride served as negative and positive controls, respectively. Following opacity and permeability measurements, the in vitro irritancy score was calculated.

Two corneas treated with the test item were clear with cloudy areas and one was slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post-treatment. The in vitro irritancy scores of the test substance-treated, negative and positive control corneas were 10.9, 4.2 and 105.5.

Therefore, the test material does not meet the criteria for classification for Severe Eye Damage Category 1 according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS), and is thus considered not to be an ocular corrosive or severe irritant in vitro but no conclusion could be made regarding eye irritating potential.

Dicalcium pyrophosphate was tested for its irritancy potential to the rabbit eye in a GLP-compliant study conducted according to OECD Guideline 405. Two New Zealand White rabbits were sequentially tested. In each case, 100 mg of the test material was placed into the conjunctival sac of one eye, the untreated eye serving as control. The treated eyes were not rinsed after exposure, and ocular effects were assessed at 1, 24, 48 and 72 hours post-instillation. No corneal effects were noted during the study. Iridial inflammation was noted in both treated eyes one hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye one hour after treatment. Minimal conjunctival irritation noted in both treated eyes at the 24-hour observation. Both treated eyes appeared normal at the 48-hour observation. The test item produced individual scores of 0/0 for corneal opacity, 0/0 for iritis, 0.3/0.3 for conjunctival redness and 0.3/0.3 for chemosis, calculated as the mean scores following gradings at 24, 48 and 72 hour after instillation. Observed effects were fully reversible within the 72 hour observation period.

Therefore, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not eye irritating.


Justification for classification or non-classification

The available data indicate that the substance does not meet the classification criteria in accordance with Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) for skin and eye irritation.