Registration Dossier

Administrative data

Description of key information

In in vivo test in rabbit indicates that the substance is moderately irritating to skin.
A BCOP test with this substance resulted to an IVIS score of 31.5.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 31 August 2011 and 04 October 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:
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:
Two New Zealand White (Hsdlf:NZW) strain rabbits were used.
At the start of the study the animals weighed 2.28 or 2.55 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
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.
Type of coverage:
semiocclusive
Preparation of test site:
other: clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
A quantity of 0.5 ml of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch.
Duration of treatment / exposure:
Up to 4 hours
Observation period:
Up to 14 days
Number of animals:
2
Details on study design:
On the day before the test each rabbit was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit. A quantity of 0.5 ml of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test item was removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated Spirits.
After consideration of the skin reactions produced in the first animal, an additional animal was treated with 0.5 ml of test item. One patch was applied to the back of the rabbit and was allowed to remain in contact with the skin for a period of four hours.
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal: 70905 Male
Time point:
other: Score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Remarks on result:
other: Loss of skin elasticity and flexibility, crust formation and glossy skin noted. Adverse reaction prevented accurate evaluation of erythema at 7 days.
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal: 709830 Male
Time point:
other: Score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Remarks on result:
other: Loss of skin elasticity and flexibility, crust formation, light brown discoloration of the epidermis, hardened light brown coloured scab and glossy skin noted. Adverse reaction prevented accurate evaluation of erythema at 7 days.
Irritation parameter:
other: Oedema Formation
Basis:
animal: 70905 Male
Time point:
other: Score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
other: Oedema Formation
Basis:
animal: 70983 Male
Time point:
other: Score at 1, 24, 48 and 72 hours, 7 and 14 days
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: Adverse reaction prevented accurate evaluation of oedema at 7 days.
Irritant / corrosive response data:
3-Minute Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 1.
Well-defined erythema and very slight oedema were noted at the treated skin site at the 24, 48 and 72 Hour observations. Crust formation, preventing accurate evaluation of erythema, was noted at the treated skin site at the 7 Day observation. Glossy skin was noted at the treated skin site at the 14 Day observation.

1-Hour Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 1.
Well-defined erythema and slight oedema were noted at the treated skin site at the 24, 48 and 72 Hour observations. Crust formation, preventing accurate evaluation of erythema, was noted at the treated skin site at the 7 Day observation. Glossy skin was noted at the treated skin site at the 14 Day observation.

4-Hour Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 2.
Well-defined erythema and slight oedema were noted at one treated skin site immediately after patch removal. Well-defined erythema and slight oedema were noted at one treated skin site with very slight erythema and slight oedema noted at the other treated skin site one hour after patch removal. Well defined erythema and slight oedema were noted at both treated skin sites at the 24, 48 and 72 Hour observations.
Loss of skin flexibility and/or elasticity was noted at both treated skin sites at the 24, 48 and 72 Hour observations. Light brown discolouration of the epidermis was also noted at one treated skin site at the 24, 48 and 72-Hour observations. Crust formation was noted at one treated skin site at the 72 Hour observation and at the other treated skin site at the 7 Day observation. Hardened light brown coloured scab was noted at one treated skin site at the 7-Day observation. Adverse reactions prevented accurate evaluation of oedema and/or erythema at both treated skin sites at the 7 Day observation. Glossy skin was noted at both treated skin sites at the 14 Day observation.
Other effects:
Bodyweight

Individual bodyweights and bodyweight changes are given in Table 3.
Both animals showed expected gain in bodyweight during the study.

Interpretation of Results

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme

The scores for erythema and oedema at the 24 and 72‑Hour readings were totalled for the two test rabbits (8 values) and this total was divided by four to give the primary irritation index of the test item. The test item was classified according to the following scheme devised by Draize J H (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of theUnited States,Austin,Texas, p.46‑59:

Primary Irritation Index

Classification of Irritancy

     0

Non-irritant

> 0 to 2

Mild irritant

> 2 to 5

Moderate irritant

> 5 to 8

Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.

The results were also interpreted according to EU labelling regulations Commission Directive 2001/59/EC and Regulation (EC) No 1272/2008 for the classification, packaging and labelling of dangerous substances.

Table 1              Individual Skin Reactions Following 3-Minute and 1-Hour Exposures

Skin Reaction

Observation Time
(following patch removal)

Individual Scores - Rabbit Number and Sex

70905Male

3-Minute Exposure

1-Hour Exposure

Erythema/Eschar Formation

Immediately

0

0

1 Hour

0

0

24 Hours

2

2

48 Hours

2

2

72 Hours

2

2

7 Days

?eCf

?eCf

14 Days

0G

0G

Oedema Formation

Immediately

0

0

1 Hour

0

0

24 Hours

1

2

48 Hours

1

2

72 Hours

1

2

7 Days

0

0

14 Days

0

0


Cf =     Crust formation

G =      Glossy skin

?e =     Adverse reaction prevented accurate evaluation of erythema

Table 2              Individual Skin Reactions Following 4-Hour Exposure

Skin Reaction

Observation Time
(following patch removal)

Individual Scores – Rabbit Number and Sex

Total

70905Male

70983Male

Erythema/Eschar Formation

Immediately

0

2

(2 )

1 Hour

1

2

( 3 )

24 Hours

2Le

2BrLeLf

4

48 Hours

2LeLf

2BrLeLf

( 4 )

72 Hours

2Le

2BrLeLfCf

4

7 Days

?eCf

?eSp

( 0-8 )

14 Days

0G

0G

( 0 )

Oedema Formation

Immediately

0

2

( 2 )

1 Hour

2

2

( 4 )

24 Hours

2

2

4

48 Hours

2

2

( 4 )

72 Hours

2

2

4

7 Days

0

?od

( 0-4 )

14 Days

0

0

( 0 )

Sum of 24 and 72-hour Readings (S)       :          16

Primary Irritation Index (S/4)                   :          16/4 = 4.0

Classification                                        :          MODERATE IRRITANT


(   ) = Total values not used for calculation of primary irritation index

Le =     Loss of skin elasticity

Lf =      Loss of skin flexibility

Br =     Light brown discolouration of the epidermis

Cf =     Crust formation

Sp =    Hardened light brown coloured scab

G =      Glossy skin

?e =     Adverse reaction prevented accurate evaluation of erythema

?od =   Adverse reaction prevented accurate evaluation of oedema

Table 3              Individual Bodyweights and Bodyweight Changes

Rabbit Number
and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 14

70905Male

2.28

2.53

0.25

70983Male

2.55

2.76

0.21


Interpretation of results:
moderately irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: Draize
Conclusions:
The test item produced a primary irritation index of 4.0 and was classified as a MODERATE IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
The test item was classified as irritant according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, the indication of danger “Irritant” and the risk phrase R 38 “IRRITATING TO SKIN” are therefore required.
Due to the persistence of inflammation, the test item was classified Category 2: Irritant according to Regulation (EC) No 1272/2008. The Signal Word “Warning” and Hazard Statement “H315: Causes skin irritation” are therefore required.
Executive summary:

Introduction. 

The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted24 April 2002)

Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No. 440/2008

Results.

3-minute and 1-hour semi-occluded applications of the test item to the intact skin of one rabbit produced no corrosive effects.

A single 4-hour, semi-occluded application of the test item to the intact skin of two rabbits produced well-defined erythema and slight oedema. Other skin reactions noted were light brown discolouration of the epidermis, loss of skin elasticity and flexibility, crust formation and a hardened light brown coloured scab. Glossy skin was noted at both treated skin sites at the 14-Day observation. No corrosive effects were noted. On occasions, adverse reactions prevented accurate evaluation of oedema and/or erythema.

Conclusion.

The test item produced a primary irritation index of 4.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.

The test item was classified as irritant according to EU labelling regulations Commission Directive 2001/59/EC. The symbol “Xi”, the indication of danger “Irritant” and the risk phrase R 38 “IRRITATING TO SKIN” are therefore required.

Due to the persistence of inflammation, the test item was classified Category 2: Irritant according to Regulation (EC) No 1272/2008. The Signal Word “Warning” and Hazard Statement “H315: Causes skin irritation” are therefore required.

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:
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 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:
31.5
Reversibility:
other:
Remarks on result:
other: See Below
Irritant / corrosive response data:
The test material induced an in vitro irritancy score of 31.5.
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 Material

31.5

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

7

3

4

15

12

10.7

1.409

1.393

 

8

3

5

15

12

10.7

1.383

1.367

 

9

2

3

14

12

10.7

1.424

1.408

 

 

 

 

 

 

10.7·

 

1.390·

31.5


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

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 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 an In Vitro Irritancy Score (IVIS).

Results

The in vitro Irritancy scores are summarised as follows:

Treatment

In Vitro Irritancy Score

Test Material

31.5

Negative Control

1.6

Positive Control

31.8

The corneas treated with the test item were clear post treatment and slightly 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 dimethylammonium C10-13-alkylbenzene sulfonate salt (MAPDA-ABS salt) is expected based on available information on the two parts of the salt ABS and 3-(methacrylamidopropyl) dimethylammonium (MAPDA). 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 of read-across attached to Chapter 13.)

 

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 was 94.1%, indicating that the substance was not irritating.

In view of the expected irritating properties and the irritating properties seen in BCOP study, as well as in in vitro EPISKIN assay with a structural analogue MAPTA-ABS salt, an in vivo confirmation study was done.

A 4-hour, semi-occluded application of the test item to the intact skin of two rabbits produced well-defined erythema and slight oedema. Other skin reactions noted were light brown discolouration of the epidermis, loss of skin elasticity and flexibility, crust formation and a hardened light brown coloured scab. Glossy skin was noted at both treated skin sites at the 14-Day observation. No corrosive effects were noted. On occasions, adverse reactions prevented accurate evaluation of oedema and/or erythema. The test item produced a primary irritation index of 4.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.

 

A BCOP test (OECD 437) with this substance resulted to an IVIS score of 31.5. 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.

There is no information on possible respiratory irritation. The likelihood for exposure via inhalation to cause irritation is very low, in view 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 vivo testing in rabbit (OECD 404) 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.