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Administrative data

Description of key information

Skin: Not irritant, EpiSkin, OECD 439, Dreher 2011

Skin: Not corrosive, EpiDerm, OECD 431, Dreher 2011

Eye: Non-corrosive / no severe eye irritant, Bovine corneas, OECD 437, Dreher 2011

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:
key study
Study period:
19 January 2011 to 16 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 431 guidelines
Qualifier:
according to
Guideline:
other: OECD 431 In Vitro Skin Corrosion: Human Skin Model Test and Method B.40 BIS of Commission Regulation EC) No 440/2008
Deviations:
no
GLP compliance:
yes
Species:
other: EpiDerm tissues (three-dimentional human skin model
Details on test animals and environmental conditions:
No test animals were used.
EpiDerm tissues were supplied by MatTek Corporation, Ashland, Massachusetts, USA and stored refrigerated. Tissues were transferred to 6-well plates with the assay medium 90 minutes before starting the assay.
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 mg
- Concentration (if solution): Undiluted test substance
Duration of treatment / exposure:
Contact time of 3 minutes or 1 hour
Observation period:
Incubated at 37°C for 3 hours
Number of animals:
No animals were used on this test.
Four EpiDerm tissues were used per test substance, negative and positive control
Details on study design:
APPLICATION OF TEST AND CONTROL MATERIALS:
Immediately prior to treatment being initiated the assay medium was replaced.
The test was performed on a total of four tissues per test article, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. Approximately 40 mg solid was placed onto the tissue and moistened with 25 µL water to ensure good contact with the tissue surface. Further tissues were treated with 40 µL distilled water (negative control) and with 40 µL 8N potassium hydroxide (positive control).
After the 3-minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material.

CELL VIABILITY MEASUREMENTS:
The assay medium was replaced with 300 µL of 1 mg/mL MTT-medium and tissues were incubated for 3 hours at 37°C. After incubation, the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight, without shaking.
The optical density of the extracted formazan was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.
Irritation / corrosion parameter:
other:
Value:
99
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. Reversibility: no data Not measured. (migrated information)
Irritation / corrosion parameter:
other:
Value:
140
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hour. Reversibility: no data Not measured. (migrated information)
Other effects / acceptance of results:
The OD values obtained for each test sample were used to calculate the percentage viability relative to the negative control, which is arbitrarily set at 100%. The prediction of corrosivity associated with the EpiDermTM model is:
1. The test substance is considered to be corrosive to skin if the viability after a three minute exposure is less than 50%, or if the viability after three minutes exposure is greater than or equal to 50% and the viability after one hour is less than 15%.
2. The test substance is considered to be non-corrosive to skin if the viability after a three minute exposure is greater than or equal to 50% and the viability after a one hour exposure is greater than or equal to 15%.
For the three minute treatment, no significant differences in tissue viability were observed following test article treatment relative to the negative control. The relative survival of tissues treated with the test article was 99% when compared to the negative control.
For the one hour treatment, no significant differences in tissue viability were observed following test article treatment relative to the negative control. The relative survival of tissues treated with the test article was 140% when compared to the negative control.
No other test substance related effects were observed.

Skin Corrosion Assay; Cell Viability Measurements

        3 minute treatment

Substance

Tissue replicate

OD570

Mean

Tissue mean

% difference between Rep’s

% Relative survival

Aliquot 1

Aliquot 2

Aliquot 3

Negative control

A

1.985

1.958

1.738

1.894

1.797

-11.3

100

B

1.693

1.716

1.695

1.701

Test article

A

1.766

1.778

1.774

1.773

1.771

-0.2

99

B

1.776

1.762

1.768

1.769

Positive control

A

0.340

0.341

0.341

0.340

0.340

0.5

19

B

0.337

0.343

0.337

0.339

 

        1 hour treatment

Substance

Tissue replicate

OD570

Mean

Tissue mean

% difference between Rep’s

% Relative survival

Aliquot 1

Aliquot 2

Aliquot 3

Negative control

A

1.732

1.725

1.697

1.718

1.641

-9.8

100

B

1.469

1.608

1.615

1.564

Test article

A

2.365

2.408

2.338

2.370

2.306

-5.8

140

B

2.212

2.257

2.254

2.241

Positive control

A

0.235

0.244

0.243

0.241

0.259

-15.0

16

B

0.273

0.279

0.279

0.277

 

Interpretation of results:
other: Not corrosive
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
The test substance was considered not to be corrosive to the in vitro skin testing model, EpiDerm.
Executive summary:

The result indicates that the substance should be non-corrosive to skin. Without further in vivo data the result has to be considered as inconclusive regarding classification.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2011 to 16 August 2011
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 439 In Vitro Skin Irritation Resconstructed Human Epidermis Test Method and Method B.46 of Commission Regulation (EC) No761/2009
Deviations:
no
GLP compliance:
yes
Species:
other: EpiSkin tissues (three-dimentional human skin model)
Strain:
other: Not applicable
Details on test animals and environmental conditions:
No test animals were used.
EpiSkin tissues were supplied by SkinEthic Laboratories, Nice, France and kept in their packaging at room temperature in a microbiological safety cabinet until use. The tissues were set up 24 hours prior to treatment by placing the tissues onto 2 mL pre-warmed maintenance medium (supplied with the EpiSkin tissues) in 12-well plates and incubating at 37°C.
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
other: Not applicable - no vehicle used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40mg
- Concentration (if solution): Undiluted test material
Duration of treatment / exposure:
15 minutes
Observation period:
42-hour recovery period
Number of animals:
No animals were used on this test.
Three EpiSkin tissues were used per test substance, negative and positive control
Details on study design:
APPLICATION OF TEST AND CONTROL MATERIALS:
Immediately prior to treatment initiation, the media under the tissues was replaced with 2.2 mL of pre-warmed assay medium (supplied with the EpiSkin tissues).
Approximately 40 mg of the test article, moistened with sterile water, was added topically to the tissue. Exposure was for 15 minutes with the test article after which, the tissues were washed using PBS and dried using cotton wool buds to remove residual test material before being transferred to a new well containing 2 mL pre-warmed maintenance medium. The tissues were then incubated at 37°C for the 42 hour recovery time period.

CELL VIABILITY MEASUREMENTS:
Upon completion of the 42 hour recovery period, the base of each tissue was rinsed with PBS before being placed on top of 2 mL of 0.3 mg/mL (final concentration) MTT in medium and incubated for three hours (37°C, 5% CO2).
After incubation, the tissues were removed from the MTT solution and their bases rinsed with PBS. The white ring was discarded and the epidermis was separated from the support layer. Both parts were placed into a sterile, sealable Eppendorf tube containing 0.5 mL acidic isopropanol. The tubes were sealed, vortexed to ensure thorough mixing and maintained at room temperature overnight protected from light.
After this extraction period, the tubes were placed in a refrigerator at 2 to 8°C for approximately 48 hours. At the end of this period, the tubes were inverted to ensure an homogenous colour. Two × 200 µL samples per tube were placed into a 96-well plate for spectrophotometric determination of optical density at 570 nm, extraction solvent was used as a blank. Tissue viability was calculated for each tissue as a percentage of the mean of the negative control tissue.
Irritation / corrosion parameter:
other:
Value:
89
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure, 42 hour recovery period. Reversibility: other: Not measured. (migrated information)
Other effects / acceptance of results:
Test substances (test articles) that reduce the viability of the epidermal tissue to below 50% of the negative control are predicted as IRRITANT (UN GHS category 2). Test substances (test articles) that do not reduce the viability of the epidermal tissue to below 50% of the negative control are predicted as NON-IRRITANT (ECVAM 2009).
The group mean viability for the test article was 89.0%.
The group mean viability for the negative control was 100%. The OD values for the negative controls were between 0.6 and 1.5, confirming appropriate performance of the assay.
The group mean viability for the positive control was 7.9%. This was in accordance with the assay acceptance criteria (≤40%) confirming appropriate performance of the assay.
These results indicate the viability of the epidermal tissue was not reduced to below 50% of the negative control and therefore may be considered to be non-irritant (ECVAM 2009). The positive control showed an appropriate response in the assay system. No other test substance related effects were observed.

Skin Irritation Assay; Cell Viability Measurements

Substance

Tissue replicate

OD570(200 µL)

Corrected mean*

Standard deviation

% Standard deviation (dose)

% Relative survival, cell viability

Aliquot 1

Aliquot 2

Tissue

Mean

Negative control

A

1.040

1.103

1.032

0.045

1.3

99.4

100.0

B

1.063

1.073

1.029

0.007

99.1

C

1.073

1.110

1.053

0.026

101.4

Test article

A

1.133

1.172

1.114

0.028

17.0

107.3

89.0

B

0.833

0.810

0.783

0.016

75.4

C

0.924

0.907

0.876

0.012

84.4

Positive control

A

0.083

0.087

0.046

0.002

3.9

4.5

7.9

B

0.107

0.124

0.077

0.012

7.4

C

0.141

0.184

0.124

0.030

11.9

Blank

0.039

0.038

 

           * = corrected for blank

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test substance was considered not to be an irritant to the in vitro skin testing model, EpiSkin.
Executive summary:

The result indicates that the substance should be non-irritating to skin. Without further in vivo data the result has to be considered as inconclusive regarding classification.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental dates: 16 February 2011 to 17 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 437 guidelines
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
Deviations:
no
GLP compliance:
yes
Species:
other: Bovine corneas
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
No test animals were used.
Bovine corneas supplied by A Traves and Son Ltd., Abattoirs, York, United Kingdom. Following slaughter the eyes were removed, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility. Upon arrival at the test facility the corneas were excised from the eyes and loaded onto specifically designed holders, filled with pre-warmed Eagle's Minimal Essential Medium (EMEM) and incubated at 32°C for 1 hour.
Vehicle:
other: No vehicle used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg
- Concentration (if solution): undiluted test material
Duration of treatment / exposure:
4 hour incubation at 32°C
Further 1.5 hours at 32°C to achieve the permeability end point
Number of animals or in vitro replicates:
No animals were used in this test.
Three corneas were used per test substance, positive and negative control
Details on study design:
APPLICATION OF TEST SUBSTANCE AND CONTROL MATERIALS:
The test article did not produce a suitable suspension or solution at a concentration of 20% w/v in 0.9% sodium chloride solution, therefore the solid test article was used as supplied. 750 mg of test article was applied to the anterior surface of each of three corneas followed by a four hour incubation at 32°C ± 1°C.
A volume of 750 µL of the negative (0.9% sodium chloride) or positive control (20% w/v Imidazole) was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.
REMOVAL OF TEST SUBSTANCE
- Washing: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red, the opacities measured and the anterior chamber emptied
- Time after start of exposure: 4 hours

SCORING SYSTEM:
In vitro Irritation Score (IVIS)

TOOL USED TO ASSESS SCORE:
For the permeability endpoint, 1 mL of sodium fluorescein (5 mg/mL solution) was added into the anterior chamber and the corneas were incubated at 32°C ± 1°C for 1.5 hours ± 5 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were transferred to a 96 well plate and analysed for optical density at 490 nanometers (OD490).
Irritation parameter:
in vitro irritation score
Value:
16.37
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The In Vitro Irritation Score (IVIS) for the test article is calculated as follows:
IVIS = mean opacity value + (15 x mean permeability value)
The test article will be concluded as a corrosive or severe irritant if it causes damage to the cornea such that the IVIS is >55.1.

The following mean in vitro irritation scores (IVIS) were calculated. The test article produced an IVIS of 16.37 and the positive control article produced an IVIS of 84.89 (within two Standard Deviations of the historical mean).
These results indicate that the IVIS for the test article was not ≥55.1 and therefore the test article may be considered not to be a corrosive or severe irritant, in compliance with the test guideline. The positive control showed an appropriate response in the assay system. The assay acceptance criteria were achieved. No other test substance related effects were observed.

Corneal Opacity

Test chemical

Cornea number

Initial opacity reading

Post incubation opacity

Change in opacity

Average change in opacity (negative control)

Corrected opacity

Mean opacity

Negative control

4

2

4

2

2.00

0.0

0.0

1

2

4

2

 

0.0

22

2

4

2

 

0.0

Positive control

20

3

73

70

N/A

68.0

60.0

6

1

59

58

 

56.0

10

1

59

58

 

56.0

Test article

24

3

20

17

N/A

15.0

16.0

21

1

18

17

 

15.0

11

2

22

20

 

18.0

Permeability

Test chemical

Cornea number

OD490reading

Corrected OD490

Mean negative control corrected OD490

Final corrected OD490

Average group corrected OD490

Negative control

4

0.050

0.005

0.005

-0.001

0.000

1

0.049

0.003

 

-0.002

22

0.053

0.008

 

0.002

Positive control

20

1.212

1.166

N/A

1.161

1.659

6

1.966

1.921

 

1.916

10

1.952

1.907

 

1.901

Test article

24

0.081

0.036

N/A

0.030

0.025

21

0.073

0.028

 

0.022

11

0.072

0.026

 

0.021

Mean blank

0.046

 

Calculated IVIS

Test chemical

Mean opacity

Mean permeability x 15

IVIS (mean opacity + (15 x mean permeability))

 

 

 

 

Negative control

0

0

0

Positive control

60

24.89

84.89

Test article

16

0.37

16.37

 

 

 

 

Interpretation of results:
other: Not corrosive or a severe irritant
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
The test subtance was considered not to cause corrosion or severe irritation to the eye under the conditions of the assay.
Executive summary:

The result indicates that the substance should be non-corrosive to the eye and not a severe eye irritant. Without furtherin vivo data the result has to be considered as inconclusive regarding classification.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an in vitro skin corrosion study conducted according to OECD 431 the test substance was found to reduce the viability of epidermal tissue by <50% and can therefore be considered as a unlikely candidate as a skin corrosive substance.

In an in vitro skin irritation study conducted according to OECD 439 the test substance was found to reduce the viability of epidermal tissue by <50% and can therefore be considered as a unlikely candidate as a skin irritating substance.

In an in vitro eye irritation study conducted according to OECD 437 the in vitro irritation score was found to be <55.1 and can therefore be considered as an unlikely candidate as an eye irritating substance.


Justification for classification or non-classification

Results from validated in vitro studies for skin corrosion and skin irritation conducted according to validated OECD guidelines 431 and 439, demonstrated the substance to be non-corrosive and non-irritating to skin. Whilst the data is only based on anin vitro study the result has to be considered inconclusive regarding classification according to (EC) 1272/2008.

Results from validated in vitro study for eye irritation conducted according to validated OECD guideline 437, demonstrated the substance is not an occular corrosive nor a severe eye irritant. Whilst the data is only based on an in vitro study the result has to be considered inconclusive regarding classification according to (EC) 1272/2008.