Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: GLP and conducted to current guideline.
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guidelines for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 2004)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: SkinEthic in vitro Reconstituted Human Epidermal (RHE) Model
Strain:
other: not applicable
Details on test animals and environmental conditions:
Not applicable.
Type of coverage:
other: not applicable.
Preparation of test site:
other: not applicable.
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg of the test material , moistened with 20 µl of sterile distilled water, was applied to the duplicate tissues.
Duration of treatment / exposure:
Duplicate SkinEthic RHE tissues were topically treated with the test material for exposure periods of 3 minutes and 60 minutes.
Observation period:
3 hour incubation period.
Number of animals:
Not applicable.
Details on study design:
TEST SITE
- Area of exposure:
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 received an equal exposure time. 20 mg of the test material, moistened with 20 µl of sterile distilled water, and 40 µl of the positive control material were also applied to the corresponding duplicate tissues, ensuring that the test material and the control materials uniformly covered the tissue surface area.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each SkinEthic RHE tissue was rinsed using Dulbecco’s phosphate buffered saline (DPBS) and placed into a ‘holding plate’. They were then transferred to an MTT ‘loading plate’ and incubated (37°C, 5% CO2) for 3 hours. At the end of this time, each SkinEthic RHE tissue was blotted dry and placed into an MTT ‘extraction plate’ to extract the reduced MTT from the tissues.
- Time after start of exposure: 3 minutes or 60 minutes.

SCORING SYSTEM:
At the end of the extraction period, the reduced MTT solution was mixed for each SkinEthic RHE tissue and 3 x 200 µl samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540nm (OD540) of each well was measured. Data are presented in the form of relative mean viability (MTT reduction in the test material treated tissues relative to negative control tissues) for each of the two exposure times.

Irritation / corrosion parameter:
other:
Value:
79.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minute exposure. Max. score: 100.0. Reversibility: other: not applicable.
Irritation / corrosion parameter:
other:
Value:
66
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minute exposure. Max. score: 100.0. Reversibility: no data not applicable.
Other effects / acceptance of results:
Direct MTT Reduction:
The absence of darkening was taken to indicate that the test material was not able to directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material:
The mean OD540 values for the negative control, test material and positive control, for both the 3 minute and 60 minute exposures are given in Table 1. The mean viabilities of the test material and positive control, relative to the corresponding negative control (i.e. same exposure time) are also given in Table 1.
The relative mean viability of the test material treated tissues was 79.3% after a 3 minute exposure and 66.0% after a 60 minute exposure.

Table 1: Mean OD540Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

Exposure Time

Mean OD5401

Relative Mean Viability
(%)

Negative Control

3 minute

1.189

100*

60 minute

1.278

100*

Positive Control

3 minute

0.145

12.2

60 minute

0.007

0.5

Test Material

3 minute

0.943

79.3

60 minute

0.843

66.0


1=     Mean of SkinEthic tissues tested in duplicate

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

Quality Criteria:

The mean OD540 of the two negative control tissues were 1.189 for the 3 minute exposure and 1.278 for the 60 minute exposure.  The negative control acceptance criterion was therefore satisfied.  

The relative mean viability of the positive control material treated tissues was 12.2% after a 3 minute exposure and 0.5% after a 60 minute exposure.

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered not to have the potential to be corrosive in vivo.
Executive summary:

Introduction: 

The purpose of this test was to evaluate the corrosivity potential of the test material using the SkinEthic in vitro Reconstituted Human Epidermal (RHE) Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the SkinEthic tissue. Cytotoxicity was determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test material treated cultures relative to the negative control. The results were used to make a prediction of the corrosivity potential of the test material. The method was designed to meet the requirements of the following:

- OECD Guidelines for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 2004)

Methods: 

Duplicate SkinEthic RHE tissues were topically treated with the test material for exposure periods of 3 minutes and 60 minutes. 

Duplicate negative control and positive control treated tissues were concurrently exposed for 3 minutes and 60 minutes.

At the end of the exposure period each SkinEthic RHE tissue was rinsed using Dulbecco’s phosphate buffered saline (DPBS) and placed into a ‘holding plate’. They were then transferred to an MTT ‘loading plate’ and incubated (37°C, 5% CO2) for 3 hours. At the end of this time, each SkinEthic RHE tissue was blotted dry and placed into an MTT ‘extraction plate’ to extract the reduced MTT from the tissues.

At the end of the extraction period, the reduced MTT solution was mixed for each SkinEthic RHE tissue and 3 x 200 µl samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540nm (OD540) of each well was measured. Data are presented in the form of relative mean viability (MTT reduction in the test material treated tissues relative to negative control tissues) for each of the two exposure times.

Results: 

The relative mean viability of the test material treated tissues was 79.3% after a 3 minute exposure and 66.0% after a 60 minute exposure.

Quality criteria: 

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

Conclusion: 

The test material was considered not to have the potential to be corrosive in vivo.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-19 to 2010-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study follows GLP and internationally accepted guideline
Qualifier:
equivalent or similar to
Guideline:
other: EU method B.46 (in vitro skin irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EPISKIN™ Reconstituted Human Epidermis model
Strain:
other: not applicable
Details on test animals and environmental conditions:
Not applicable
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg


VEHICLE
Test material was used as supplied
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
Not applicable
Details on study design:
APPLICATION OF TEST MATERIAL
- Area of exposure: The test material was applied topically to the reconstituted epidermis ensuring uniform coverage. The epidermis surface had been moistened with 5 µL of sterile distilled water to improve contact between the solid test material and the epidermis.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each tissue was rinsed with a Phosphate Buffered Saline solution containing Ca2+ and Mg2+ before incubating for approximately 42 hours at 37 °C in 5% CO2 air
- Time after start of exposure: 15 minutes


SCORING SYSTEM:
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-labelled 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-labelled 96-well plate. The optical density was measured at 540 nm.
Irritation / corrosion parameter:
other:
Value:
>= 107 - <= 110.6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Please refer to table 1 for tabulated results including control.
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues was 108.2 % after a 15-minute exposure.

QUANTITATIVE MTT ASSESSMENT (percentage tissue viability):For the test material, the relative mean tissue viabilities were compared to the mean of the negative control treated tissues (n = 3). The relative mean viabilities were calculated in the following way:

% Relative mean viability = (mean OD540 of test material/mean OD540 of negative control) x 100
The test material was found not to directly reduce MTT.

Table 1: Mean OD540 values and % viabilities for the negative control material, positive control material and test material

Material

OD540of tissues

Mean OD540of triplicate tissues

± SD of OD540

Relative individual tissue viability %

Relative mean % viability

± SD of % viability

Negative control material

0.827

 

0.833

 

0.040

99.3

 

100*

 

4.7

0.776

95.6

0.875

105.0

Positive control material

0.065

 

0.054

 

0.010

7.8

 

6.5

 

1.2

0.052

6.2

0.045

5.4

Test material

0.611

 

0.505

 

0.092

73.3

 

60.6

 

11.1

0.458

55.0

0.445

53.4

SD = Standard deviation * = The mean viability of the negative control tissues is set at 100%.

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative control Material

-

-

-

Positive Control Material

++

++

++

Test Material

-

-

-

MTT visual scoring scheme:

-         - = blue tissue (viable)

-         + = blue/white tissue (semi-viable)

-         ++ = tissue is completely white (dead)

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied, i.e. positive control and negative control acceptance criteria.

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: other: Expert judgement based on the criteria set out in the study protocol
Conclusions:
The test material was considered to be a non-irritant to the reconstituted human epidermis model EPISKIN™.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-09 to 2010-02-23.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and conducted to current guideline.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Three New Zealand White rabbits were supplied by Harlan Laboratories UK Ltd, Hillcrest, Belton, Loughborough, UK.
- Age at study initiation: Twelve to twenty weeks old.
- Weight at study initiation: 2.69 to 2.83 kg
- Housing: The animals were individually housed in suspended cages.
- Diet : Free access to food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.
- Water : Free access to mains drinking water was allowed throughout the study.
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve limits of 17 to 23°C.
- Humidity (%): Set to achieve limits of 30 to 70%
- Air changes (per hr): At least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test material, moistened sufficiently with 0.5 ml of distilled water to achieve a paste.


Duration of treatment / exposure:
Animal were exposed for 4 hours.
Observation period:
Animals were observed for 72 hours for skin reactions.
Number of animals:
3.
Details on study design:
TEST SITE
- Area of exposure: A quantity of 0.5 g of the test material, moistened sufficiently with 0.5 ml of distilled water to achieve a paste, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch.
- Type of wrap if used: The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Any residual test material was removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: Four hours after application.

SCORING SYSTEM:
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale (see below):
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
(Male 68853)
Time point:
24/48/72 h
Score:
ca. 2
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Remarks on result:
other: Adverse reaction prevented accurate evalution at 48 and 72 hour time points and therefore the mean score is not able to be accurately calculated.
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
(Male 68854)
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
(male 68855)
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
(male 68853)
Time point:
24/48/72 h
Score:
ca. 3
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Remarks on result:
other: Adverse reaction prevented accurate evaluation at 48 and 72 hour time points and therefore the mean score is not able to be accurately calculated.
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
(male 68854)
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
(male 68855)
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritant / corrosive response data:
The individual scores for erythema/eschar and oedema are given in Table 1 (see any other information on results section).

Well-defined erythema and slight to moderate oedema were noted at all treated skin sites immediately and one hour after patch removal and at the 24 hour observation. Well defined erythema and very slight oedema were noted at two treated skin sites at the 48 and 72-hour observations.

An area of haemorrhage, approximately 5 mm x 4 mm in size, was noted in the centre of one treated skin site immediately and one hour after patch removal and at the 24 hour observation. Blanching surrounding the haemorrhage was also noted one hour after patch removal and at the 24-hour observation. Hardened dark brown/black coloured scab, preventing accurate evaluation of erythema and oedema, was noted at one treated skin site at the 48, 72-hour, 7 and 14-day observations. Light brown discolouration of the epidermis was noted at two treated skin sites at the 24, 48 and 72 hour observations with slight desquamation noted at the 7-day observation.

Two treated skin sites appeared normal at the 14-day observation.

Other effects:
Bodyweight:
Individual bodyweights and bodyweight changes are given in Table 2 (see any other information on results section).
All animals showed expected gain in bodyweight during the study.

Measurement of pH

The pH of the test material was determined prior to commencement of the study and found to be as follows:

Preparation

pH Measurement

immediately

after 10 minutes

10% w/w aqueous preparation of the test material

6.1

6.2

Table 1 Individual Skin Reactions

Skin Reaction

Observation Time
(following patch removal)

Individual Scores – Rabbit Number and Sex

68853Male

68854Male

68855Male

Erythema/Eschar Formation

Immediately

2Hd

2

2

1 Hour

2HdBl

2

2

24 Hours

2HdBl

2Br

2Br

48 Hours

?eSt

2Br

2Br

72 Hours

?eSt

2Br

2Br

7 Days

?eSt

0D

0D

14 Days

?eSt

0

0

Oedema Formation

Immediately

2

2

2

1 Hour

2

2

2

24 Hours

3

2

2

48 Hours

?od

1

1

72 Hours

?od

1

1

7 Days

?od

0

0

14 Days

?od

0

0


Hd=    Area of haemorrhage approximately 5 mm x 4 mm in centre of the test site

Bl =     Blanching surrounding the area of haemorrhage

Br =     Light brown discolouration of the epidermis

D =      Slight desquamation

St =     Hardened dark brown/black coloured scab

?e =    Adverse reaction prevents accurate evaluation of erythema

?od =  Adverse reaction prevents accurate evaluation of oedema

Table 2              Individual Bodyweights and Bodyweight Changes

Rabbit Number
and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 14

68853Male

2.78

2.98

0.20

68854Male

2.69

2.94

0.25

68855Male

2.83

3.19

0.36

Interpretation of results:
Category 1C (corrosive) based on GHS criteria
Conclusions:
The test material is classified as corrosive (category 1C ) under GHS.

The test material was also classified as corrosive to rabbit skin according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “C”, the indication of danger “Corrosive” and the risk phrase R 34 “CAUSES BURNS” are therefore required.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

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:
supporting study
Study period:
2010-01-26 to 2010-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP. Study follows an appropriate protocol for in vitro eye irritation potential without any deviations from the study design.
Qualifier:
no guideline available
Principles of method if other than guideline:
The aim of the study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.
GLP compliance:
yes (incl. certificate)
Species:
other: Reconstituted Corneal Epithelium
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST CULTURE
- Source: SkinEthic Laboratories, Nice, France
- Date Received : 26/01/2010
- Culture details: Day 6 cultures
- Storage of culture: Cultures were stored at room temperature on arrival and then transferred into 24-well plates containing 300 µL of maintenance medium. No air bubbles were present under the tissue inserts. Tissues were incubated overnight at 37 °C, 5% CO2 in air.
Tissue preparation: Using sterile techniques, 1 mL of maintenance medium at room temperature dispensed into the required number of wells in a 6-well plate. Each well was labelled with the details of treatment and the appropriate exposure time. Separate treatment plates were used for the test material and controls (negative and positive) to avoid cross contamination. Before treatment, 7 day old cultures were transferred into the treatment plates containing maintenance medium.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied : Tissues were treated with 30 mg of the test material.

VEHICLE
Test material was used as supplied

CONTROLS:
- Amount(s) applied: 30 µL of Solution A was applied as a negative control and 30 µL of SDS 1.0% (w/v) as a positive control. Solution A was comprised of Na2HPO4 0.142 g/L, Glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L.
Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile distilled water.
Duration of treatment / exposure:
Cultures were exposed for 10 minutes to the test material.
Observation period (in vivo):
Skin cultures were examined after three hours.
Number of animals or in vitro replicates:
All test substances were tested in triplicate (including controls)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Cultures were rinsed by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated "holding plate" containing 300 µL of maintenance medium (at room temperature) until all tissues were rinsed..
- Time after start of exposure: 10 minutes

STAINING PROCEDURE:
Staining: After rinsing, the tissues (two per group) were transferred into a pre-labelled 24-well plate containing 300 µL of a 0.5 mg/mL MTT solution prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37 °C, 5 % CO2 in air.


SCORING SYSTEM:

- Tissue viability (OD): After incubation with MTT, the tissues were visually examined and the degree of MTT staining was evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT and transferred to a pre-labelled 24-well plate containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

- Histology: If deemed necessary, the histopathology of the remaining insert was examined. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded for each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as blanks. The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using Anthos 2001 microplate reader. One tissue for each treatment groups was retained for assessment of tissue histopathology. Tissues were cut out of the polycarbonate inserts with a sharp scalpel. The tissues were cut in half. Both halves were placed into a pre-labelled 1.5 mL Eppendorf tube containing 1 mL of 10% Formalin and stored at room temperature.
To determine histopathological changes the tissues are observed for any changes in thickness or organisation of the cells. The negative control tissues should have a constant thickness devoid of terminally differentiated cell, and feature a regular and compact shape. Cells must maintain attachment via multiple desmosomes. Positive control tissues should have a disintegration of most of the upper cell layers of the epithelial tissue. The remaining basal cells should be loosely attached to the polycarbonate substratum.

-Interpretation of data: Quantitative MTT Assessment (percentage of viable tissue)
The relative mean tissue viabilities are compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities are calculated using the following: (mean OD540 of test material/mean OD540 of negative control) x 100

TOOL USED TO ASSESS SCORE: Anthos 2001 microplate reader.
Irritation parameter:
other: Mean tissue viability
Run / experiment:
Mean
Value:
> 0.187 - < 0.278
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
The relative mean viability of the test material treated tissues after a 10 minute exposure was 62.4%.

-Interpretation of data: Quantitative MTT Assessment (percentage of viable tissue)
The relative mean tissue viabilities are compared to the mean of the untreated negative control tissues (n = 2). The relative mean viabilities are calculated using the following: (mean OD540 of test material/mean OD540 of negative control) x 100
Other effects:
The test material was found to not directly reduce MMT. It was deemed unnecessary to examine tissue histopathology.

Table 1: Assessment of Eye Irritation Potential – Viability of RHC Tissues

 

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative control

0.911

0.954

100*

0.996

Positive control

0.312

0.333

34.9

0.353

Test material

0.278

0.233

24.4

0.187

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

 

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

 

Material

Score

Tissue 1

Tissue 2

Negative control

-

-

Positive control

+

+

Test material

+

+

- = Blue tissue (viable)

+ = Blue/White tissue (semi viable)

++ = Tissue completely white (dead)

Interpretation of results:
irritating
Conclusions:
According to the protocol followed, the test material was found to be an irritant to the reconstituted human corneal epithelial model, SkinEthic.
Endpoint:
eye irritation
Remarks:
other: ex-vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and conducted to guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The neat test material was applied for 240 minutes. Negative and positive control materials were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS)
GLP compliance:
yes (incl. certificate)
Species:
other: Adult Bovine eyes sourced from local abattoir as a by-product of freshly slaughtered animals.
Strain:
other: Cattle
Details on test animals or tissues and environmental conditions:
Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: no control animals were used, but three bovine eyes were used for both the positive and negative controls
Amount / concentration applied:
Not reported
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
No animals were used, but three bovine eyes were used.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period the neat test material and control materials 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.
- Time after start of exposure: 4 hours

SCORING SYSTEM:

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 from each 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 corneaThe 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 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 material induced a response through only one of the two endpoints


TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
overall irritation score
Basis:
other: mean opacity value + (15 x mean OD492 value)
Time point:
other: 4 hours
Score:
82.7
Reversibility:
not specified
Irritant / corrosive response data:
Not reported
Other effects:
Not reported

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 from each 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 OD492was calculated by subtracting the mean OD492of the negative control corneas from the OD492value of each treated cornea. The OD492value of each treatment group was calculated by averaging the corrected OD492values of the treated corneas for the treatment group.

In vitro irritancy score:

The following formula was used to determine the in vitro score:

           In VitroIrritancy Score = mean opacity value + (15 x mean OD492value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test material 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.

Visual observation

The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement

Data interpretation

A test material that induces an in vitro irritancy score >= 55.1 is defined as an ocular corrosive or severe irritant.

                  

Corneal opacity and permeability measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1. 

                      

Corneal epithelium condition

The condition of the cornea immediately after rinsing is given in Table 2.

The corneas treated with the test material or positive control material were cloudy post treatment. The corneas treated with the negative control material were clear post treatment.

 In Vitro Irritancy Score

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Classification

Test Material

82.7

severe irritant

Negative Control

1.5

mild irritant

Positive Control

119.8

severe irritant

Table1              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

1

2

1

 

0.026

 

 

2

2

3

1

 

0.027

 

 

3

1

2

1

 

0.039

 

 

 

 

 

1.0*

 

0.031¨

 

1.5

Positive Control

7

1

74

73

72.0

3.425

3.394

 

8

1

74

73

72.0

2.490

2.459

 

9

1

71

70

69.0

3.940

3.909

 

 

 

 

 

71.0·

 

3.254·

119.8

Test Material

4

1

76

75

74.0

0.336

0.305

 

5

1

80

79

78.0

0.303

0.242

 

6

2

87

85

84.0

0.257

0.226

 

 

 

 

 

78.7·

 

0.268·

82.7


OD= Optical density                 * = Mean of the post treatment-pre‑treatment values      ¨= Mean permeability             ·= Mean corrected value

Table2              Corneal Epithelium Condition

Treatment

Cornea Number

Observation

Immediately after Rinsing

Negative Control

1

clear

2

clear

3

clear

Positive Control

7

cloudy

8

cloudy

9

cloudy

Test Material

4

cloudy

5

cloudy

6

cloudy

Interpretation of results:
corrosive
Conclusions:
The test material is corrosive to the eyes and requires classification as R41 and Cat 1 eye irritant.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

Manganese dinitrate is classified as corrosive to the skin based on in vivo rabbit study (IUCLID section 7.3.1). It is classified as corrosive to the eye based on the validated BCOP method (IUCLID section 7.3.2).