Registration Dossier

Administrative data

Description of key information

Skin irritation:
The test substance was assessed for skin irritation according to OECD Guideline 439 and EU Method B46. The test substance was considered as a skin irritant.
The test substance was also assessed for primary skin irritation in rabbits according to FDA standards. Based on the mean irritation scores, the test substance was considered a severe skin irritant.
Eye irritation:
The test substance was assessed for eye irritation according to OECD Guideline 437 using excised bovine corneas. The test substance was not considered to be an ocular corrosive or severe irritant.
The test susbtance was also assessed for eye irritation in rabbits according to FDA standards. The test substance showed slight irritation insufficient for classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 22 August 2012 and 27 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 439
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human epidermis model
Strain:
other: reconstructed human epidermis model
Details on test animals and environmental conditions:
Not applicable
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: Not applicable
Controls:
no
Amount / concentration applied:
TEST ITEM

The test item was used as supplied.

Amount(s) applied (volume or weight with unit):
10 µl of the test item was applied to the epidermis surface.

Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
Area of exposure:
10 µl of the test item was applied to the epidermis surface.

PERCENTAGE COVERAGE:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

EXPOSURE TIME:
15 Minutes post exposure

TEST ITEM REMOVAL:
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.


SCORING SYSTEM
Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant

Mean tissue viability is >50% : Non-Irritant

Irritation / corrosion parameter:
other:
Remarks on result:
other:
Remarks:
Basis: mean Viability of cells. Time point: Day 6. Reversibility: other: Not applicable. Remarks: See relative mean viability below.. (migrated information)
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 19.5% after a 15 Minute exposure period.
Other effects:
No

RESULTS

Direct MTT Reduction

The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT.

Test Item, Positive Control Item and Negative Control Item

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 viabilities 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 was19.5% after a 15-Minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was10.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 3.1%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.686 and the standard deviation value of the percentage viability was 2.0%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 8.5%. The test item acceptance criterion was therefore satisfied.

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

Item

OD540 of tissues
tvt

OD540 of tissues
corrected for direct reduction of MTT (0.031)

Mean of OD540
±SD

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of percentage viability

Negative Control

0.684

 

0.686
±0.014

99.7

100*

±2.0

0.674

98.3

0.701

102.2

Positive Control

0.098

 

0.074
±0.021

14.3

10.8

±3.1

0.065

9.5

0.059

8.6

Test Item

0.098

0.067

0.133
±0.058

9.8

19.5

±8.5

0.207

0.176

25.7

0.188

0.157

22.9

Corrected viability of treated killed tissues

=

0.109 (tkt)-0.078 (ukt) = 0.031


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

OD540       =         optical density

SD     =         Standard deviation

tvt      =         treated viable tissues

tkt       =         treated killed tissues

ukt      =         untreated killed tissues

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The test item was classified as irritant. The following classification criteria apply:
EU DSD (67/548/EEC) Irritant requires symbol “Xi” risk phrase R38 “Irritating to Skin”.
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.
Executive summary:
Introduction: The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 -Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-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.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results: The relative mean viability of the test item treated tissues was19.5% after the 15-Minute exposure period.

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

Conclusion: The test item was classified as irritant. The following classification criteria apply:

EU DSD (67/548/EEC) Irritant requires symbol “Xi” risk phrase R38 “Irritating to Skin”.
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
other: ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was performed on 09 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD 437
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Excised Bovine Cornea
Strain:
other: Not Applicable
Details on test animals or tissues and environmental conditions:
Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST ITEM

-Amounts(s) applied (volume or weight with unit):
0.75 mL of the test item was applied to triplicate corneas.

-Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
TEST SITE
-Area of exposure
0.75 mL of the test item was applied to triplicate corneas.

-% coverage:
The test item was topically applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

-Type of wrap used:
None used

REMOVAL OF TEST ITEM
-Washing (if done):
At the end of the exposure period the test item was removed from the anterior chamber and each cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenaol red.

-Time after start of exposure:
10 minutes post exposure

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 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 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.

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 greater than or equal to 55.1 is defined as an ocular corrosive or severe irritant.



Irritation parameter:
cornea opacity score
Basis:
other: Mean Score of Opacity & Permeability
Time point:
other: 120 Minutes Post Rinsing
Max. score:
11.6
Reversibility:
other:
Remarks on result:
other: See Below
Irritant / corrosive response data:
The test item induced an in vitro irritancy score of 11.6
Other effects:
The corneas treated with the test item were clear post treatment and slightly cloudy post incubation.

RESULTS

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 and at the final opacity measurement is given in Table 2.

The corneas treated with the test item were clear post treatment and slightly cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In Vitro Irritancy Score

The results are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Item

11.6

Negative Control

1.6

Positive Control

35.1

Criteria for an Acceptable Test

The positive control In Vitro irritancy Score was within the range of 30.9 to 67.7. The positive 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-Incubation

Post-Incubation-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

2

1

2

0

 

0.033

 

 

2

2

2

4

2

 

0.034

 

 

3

5

4

6

1

 

0.043

 

 

 

 

 

 

1.0*

 

0.037 +

 

1.6

Positive Control

4

2

26

24

22

21.0

1.130

1.093

 

5

1

24

22

21

20.0

0.989

0.952

 

6

2

28

24

22

21.0

0.876

0.839

 

 

 

 

 

 

20.7·

 

0.962·

35.1

Test Item

7

1

4

14

13

12.0

0.101

0.064

 

8

2

5

14

12

11.0

0.170

0.133

 

9

1

2

10

9

8.0

0.088

0.051

 

 

 

 

 

 

10.3·

 

0.083·

11.6


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


Table 2          Corneal Epithelium Condition

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

1

clear

clear

2

clear

clear

3

clear

clear

Positive Control

4

cloudy

cloudy

5

cloudy

cloudy

6

cloudy

cloudy

Test Item

7

clear

slightly cloudy

8

clear

slightly cloudy

9

clear

slightly cloudy


Interpretation of results:
other: Non-Corrosive
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
The test item was considered not to be an ocular corrosive or severe irritant.
Executive summary:

Introduction. A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”

Method. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Results. The in vitro Irritancy scores are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Item

11.6

Negative Control

1.6

Positive Control

35.1

Conclusion. The test item was considered not to be an ocular corrosive or severe irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

The test substance was assessed for skin irritation according to OECD Guideline 439 and EU Method B46. The test substance was considered as a skin irritant.

The test substance was also assessed for primary skin irritation in rabbits according to FDA standards. Based on the mean irritation scores, the test substance was considered a severe skin irritant.

Eye irritation:

The test substance was assessed for eye irritation according to OECD Guideline 437 using excised bovine corneas. The test substance was not considered to be an ocular corrosive or severe irritant.

The test susbtance was also assessed for eye irritation in rabbits according to FDA standards. The test substance showed slight irritation insufficient for classification.


Justification for selection of skin irritation / corrosion endpoint:
The target substance was assessed according to OECD guidelines.

Justification for selection of eye irritation endpoint:
The target substance was assessed according to OECD guidelines.

Effects on skin irritation/corrosion: slightly irritating

Justification for classification or non-classification

The test substance was assessed for skin and eye irritation. In accordance with Regulation 67/548/EEC (DSD) and Regulation (EC) No. 1272/2008 (CLP), the test substance is classified as a skin irritant.

There was no potential for eye irritation.