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Description of key information

In in vitro testing in reconstituted human epidermis model (OECD 439/EU B46) the viability following a treatment period of 15 minutes and a post-exposure incubation period of 42 hours resulted to 39.6%. As this is below 50% this indicates that the substance is irritating to skin.
A BCOP test with this substance resulted to an IVIS score of 36.3.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 26 July 2011 and 01 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 439
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals or test system and environmental conditions:
Not applicable
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

The test material was applied neat.

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

Concentration (if solution):
The test material 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:
Approximately 10 µl of the test material was applied to the epidermis surface.

% coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

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

Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability):
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

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 (Xi) R38

Mean tissue viability is >50% : Non-Irritant
Irritation / corrosion parameter:
other: other: Viability of cells
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 material treated tissues was 39.6% after a 15 minute exposure.
Other effects:
No

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1(see below). The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1(see below).

The relative mean viability of the test material treated tissues was 39.6% after a 15-Minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤18%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤18%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was ≤18%. 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

Item

OD540of tissues

Mean OD540of triplicate tissues

±SDof OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.654

0.660

0.040

99.1

100*

6.1

0.623

94.4

0.703

106.5

Positive Control Item

0.060

0.049

0.011

9.1

7.4

1.7

0.038

5.8

0.049

7.4

Test Item

0.304

0.261

0.044

46.1

39.6

6.6

0.263

39.8

0.217

32.9

SD=    Standard deviation

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

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The test item was considered to be Irritant (I) EU Risk Phrase R38.
Executive summary:
Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted 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 reconstituted human epidermal cultures following topical exposure to the test material 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 material 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 materials 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.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 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 material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 39.6% after a 15-minute exposure.

Quality criteria: 

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

Conclusion:  The test item was considered to be Irritant (I) EU Risk Phrase R38.
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:
key study
Study period:
The study was performed on 13 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
other: OECD 437
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 MATERIAL

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

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

VEHICLE
No vehicle used
Duration of treatment / exposure:
The undiluted test material 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 material was applied to triplicate corneas.

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

-Type of wrap used:
None used

REMOVAL OF TEST SUBSTANCE
-Washing (if done):
At the end of the exposure period the test material 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.

-Time after start of exposure:
10 minutes post exposure

SCORING SYSTEM:
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.

The following formula was used to determine the in vitro score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

DATA INTERPRETATION
A test material that induces an in vitro irritancy score > than or equal to 55.1 is defined as an ocular corrosive or severe irritant.

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.
Irritation parameter:
cornea opacity score
Basis:
other: Mean Score of Opacity & Permeability
Time point:
other: 120 Minutes Post Rinsing
Max. score:
36.3
Reversibility:
other:
Remarks on result:
other: See Below
Irritant / corrosive response data:
The test material induced an in vitro irritancy score of 36.3.
Other effects:
The corneas treated with the test item were clear post treatment and 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 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 Material

36.3

Negative Control

1.6

Positive Control

31.8

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

3

4

2

 

0.020

 

 

2

3

3

4

1

 

0.014

 

 

3

3

3

4

1

 

0.013

 

 

 

 

 

 

1.3*

 

0.016#

 

1.6

Positive Control

4

4

33

29

25

23.7

0.729

0.713

 

5

3

21

20

17

15.7

0.793

0.777

 

6

3

31

25

22

20.7

0.881

0.865

 

 

 

 

 

 

20.0·

 

0.785·

31.8

Test Item

10

2

12

22

20

18.7

1.000

0.984

 

11

2

9

21

19

17.7

1.037

1.021

 

12

3

13

27

24

22.7

1.338

1.322

 

 

 

 

 

 

19.7·

 

1.109·

36.3


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

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

cloudy

8

clear

cloudy

9

clear

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 material to the isolated bovine cornea. The method was designed to meet the requirements of the following:

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

Method. 

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 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 anIn VitroIrritancy Score (IVIS).

Results. 

Thein vitroIrritancy scores are summarised as follows:

Treatment

In VitroIrritancy Score

Test Material

36.3

Negative Control

1.6

Positive Control

31.8

The corneas treated with the test item were clear post treatment and cloudy post incubation.

 

Conclusion. 

The test item was considered not to be an ocular corrosive or severe irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Irritation for(3-(methacryloylamino)propyl) trimethylammonium C10-13-alkylbenzene sulfonate salt (MAPTA-ABS salt) is expectedbased on available information on two parts of the salt ABS and 3-(methacrylamidopropyl) trimethylammonium (MAPTA), as ABS is (severely) irritating to skin and eyes and also for the methacrylamide part there is indication of possible skin irritation and severe eye irritation. (See document in support ofread-across attached to Chapter 13.)

Confirmation was obtained by testing the substance in in vitro test system for skin irritation EPISKIN reconstituted human epidermis model (OECD 439/EU B46). After a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours, viability was measured by MTT reduction, and resulted to 39.6%. As viability is below ≤ 50% this indicates that the substance is irritating.

A BCOP test (OECD 437) with this substance resulted to an IVIS score of 36.3. This score indicates that the substance is not corrosive to the eyes. On the other hand this score is indicative for possible irritation, which again is also expected on the bases of the information on the two separate ABS and methacrylamide parts of the salt. On the basis of this information it was decided that no furtherin vivotesting is needed as corrosion or non-irritation are both not conceivable results. Respiratory irritation:

There is no information on possible respiratory irritation. However, the likelihood for exposure via inhalation to cause irritation, is very low, inview of the high boiling point (> 300 °C) and very low vapour pressure (< 0.00000084 Pa at 20°C). Based on the available information on skin and eye irritation, there is theoretically some concern in this respect only in case of exposures to aerosols.


Justification for selection of skin irritation / corrosion endpoint:
Only study available.

Justification for selection of eye irritation endpoint:
Only study available.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Test results from in vitro testing in a reconstituted human epidermis model (OECD 439/EU B46) indicate that the substance should be classified as Cat.2 skin irritation under GHS.

Test results from BCOP study (OECD 437) also confirmed that the substance should be classification for eye irritation Cat.2.