Registration Dossier

Administrative data

Description of key information

The potential for the substance, the Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5nonylphenyl)methyl]-N-methylaminoacetate to cause skin irritation and corrosion has been investigated in studies conducted in vitro. In an in vitro skin irritation study using the EPISKIN Reconstructed Human EpiDermis Model, conducted according to OECD TG 439, the substance was determined to be “non-irritant.” Furthermore, the substance was determined to be non-corrosive to skin in the in vitro EPIDERM Skin Corrosion Test conducted according to OECD TG 431.

In an in vitro study to assess the potential for the substance to cause eye irritation and damage using the Bovine Corneal Opacity and Permeability (BCOP) conducted according to OECD TG 437, inconclusive results were obtained. In the assay, vitro irritancy score (IVIS) obtained for the substance was 12.5. Since the score was not > 55, the substance was not determined to have the potential to cause serious eye irritation or damage. On the basis that the score was >3 and <=55, no prediction of eye irritation potential of the substance can be made. Taking into consideration that the substance is neither corrosive nor irritating to the skin, and that the IVIS score is towards the lower end of the range, in a precautionary approach, a classification for eye irritation in Category 2 (Causes serious eye irritation; H319) has been assigned to the substance according to Regulation (EC) 1272/2008. Further investigations of the eye irritation potential of the substance are on-going.  

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 16 January 2019 Experimental Completion Date: 18 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
other:
Cell source:
other:
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25mg
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 Minute
Value:
>= 94
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 Minute
Value:
>= 58.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 Minute

100

3.8

94.0

60 Minute

100

3.1

58.6

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

 

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

 

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

Methods

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

 

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

 

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 viabilities for each treatment group were as follows:

 

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 Minute

100

3.8

94.0

60 Minute

100

3.1

58.6

 

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

 

Conclusion

 

The test item was considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 06 February 2019 Experimental Completion Date: 11 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
other:
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Approx. 10 mg
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 (i.e. triplicate tissues)
Irritation / corrosion parameter:
% tissue viability
Value:
>= 105.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The relative mean viability of the test item treated tissues was 105.4% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

Item

OD570of tissues

Mean OD570of triplicate tissues

±SD of OD570

Relative individual tissue viability (%)

Relative mean viability (%)

±SD of relative mean viability (%)

Negative Control

0.818

0.854

0.037

95.8

100*

4.3

0.892

104.4

0.851

99.6

Positive Control

0.044

0.033

0.010

5.2

3.9

1.2

0.026

3.0

0.028

3.3

Test Item

1.080

0.900

0.189

126.5

105.4

22.2

0.918

107.5

0.703

82.3

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

Introduction

 

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTMreconstructed 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 colorimetric 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/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

Methods

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

 

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200uL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

 

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 was 105.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

 

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

 

Conclusion

 

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

 

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 cannot be determined).

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 Start Date: 21 February 2019 Experimental Completion Date: 21 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
09 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3 eyes per exposure (i.e. negative control, positive control and test item)
Irritation parameter:
cornea opacity score
Value:
>= 6
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
>= 12.7
Negative controls validity:
valid
Positive controls validity:
valid

Treatment

Cornea Number

Opacity

Permeability (OD492)

In VitroIrritancy Score

Pre-Treatment

Post Treatment

Post Incubation

Post Incubation – Pre Treatment

Corrected Value

 

Corrected Value

Negative Control

1

4

3

4

0

 

0.003

 

 

2

2

2

2

0

 

0.001

 

 

3

3

2

2

0

 

0.000

 

 

 

 

 

 

0.0*

 

0.001$

 

0.0

Positive Control

6

3

30

31

28

28.0

0.849

0.848

 

9

3

29

34

31

31.0

1.250

1.249

 

10

3

26

27

24

24.0

0.998

0.997

 

 

 

 

 

 

27.7^

 

1.031^

43.1

Test Item

11

3

7

11

8

8.0

0.656

0.655

 

12

3

7

8

5

5.0

0.359

0.358

 

14

2

7

7

5

5.0

0.319

0.318

 

 

 

 

 

 

6.0^

 

0.443^

12.7

OD = Optical Density

*= Mean of the post-incubation – pre-treatment values

^= Mean corrected value

$=Mean permeability

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

Introduction

 

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

 

Methods

 

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 anIn VitroIrritancy Score (IVIS).

 

Data Interpretation

 

The test item is classified according to the prediction model as follows:

 

IVIS

UN GHS

≤ 3

No category

> 3; ≤ 55

No prediction can be mae

> 55

Category 1

 

Results

 

TheIn Vitroirritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test Item

12.7

Negative Control

0.0

Positive Control

43.1

 

Conclusion

 

No prediction of eye irritation can be made.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation and Corrosion

The potential for the substance, the Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5nonylphenyl)methyl]-N-methylaminoacetate to cause skin irritation was investigated in an in vitro skin irritation study using the EPISKIN Reconstructed Human EpiDermis Model, conducted according to OECD TG 439. In the study, triplicate reconstructed human epidermal tissue cultures were treated topically with the substance for an exposure period of 15 minutes, followed by a post-exposure incubation period of 42 hours.  The cytotoxicity of the substance in the cultures was assessed using a colorometric MTT reduction assay in which the viability of cells was measured by the enzyme reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The relative mean viability of the treated tissues was 105.4% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

The quality criteria required for acceptance of results in the test were not fully satisfied; this was reported as a deviation but was not considered to have affected the integrity or validity of the study. The standard deviation value of the % viability from the three test item treated tissues (22.2%) marginally exceeded the upper limit of the assay acceptance criteria (≤18%).

Based on the results of the study, the substance was determined to be “non-irritant.”

The potential for the substance to cause skin corrosion was investigate in the in vitro EPIDERM Skin Corrosion Test conducted according to OECD TG 431. In the assay, the corrosive potential of the substance was assessed based on cytotoxicity in duplicate EpiDerm human skin tissues following exposure periods of 3 and 60 minutes. Cytotoxicity was measured by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The percentage relative mean viabilities of the substance following 3 and 60 minutes exposure periods were 94 and 58.6 % respectively. The quality criteria required for acceptance of results in the test were satisfied

Based on the findings of the study, the substance was determined to be non-corrosive to skin.

Eye Irritation

A BCOP (OECD 437) study conducted with the test substance was inconclusive and consequently, no prediction with respect to eye irritation potential can be made from the result. The in vitro irritation score (IVIS) was calculated to be 12.7 - which is on the lower end of the scale with respect to eye irritation (i.e. for a test substance to be irritant, it must have an IVIS greater or equal to 55).

The potential for the substance to cause eye irritation and damage was investigated in vitro using the Bovine Corneal Opacity and Permeability (BCOP) assay in a study conducted according to OECD TG 437. In the assay, the undiluted substance was applied for 10 minutes to bovine cornea, following by an incubation period of 120 minutes.  Negative and positive control items were tested concurrently.  The two endpoints assessed in the assay, 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).

The BCOP enables a differentiation to be made between test item with the potential to cause serious eye irritation and damage from those that lack irritation potential. To this end,  an IVIS <=3 indicates that a test item can be regarded as unclassified for eye irritation, whereas a test item that gives an IVIS > 55 is regarded as having the potential to cause serious eye damage, necessitating classification in Category 1 in respect of this endpoint according to Regulation (EC) 1272/2008. No conclusive prediction of eye irritation potential is possible when the IVIS is >3 and <=55.

The in vitro irritancy score (IVIS) obtained for the substance was 12.5. On the basis that the score was >3 and <=55, the substance was not determined to have the potential to cause serious eye irritation or damage or to conclusively lack eye irritation hence no prediction of the eye irritation potential of the substance can be made, and the results from the study are inconclusive. Taking into consideration that the substance is neither corrosive nor irritating to the skin, and that the IVIS score is towards the lower end of the range (3 to 55 for which no prediction can be made), in a precautionary approach, a classification for eye irritation in Category 2 has been assigned to the substance according to Regulation (EC) 1272/2008. Further investigations are on-going to characterise the eye irritation properties of the substance.

Justification for classification or non-classification

Skin Irritation and Corrosion

The substance was not found to be irritating or corrosive to the skin in studies in vitro. On this basis, the substance is deemed to lack skin irritation potential and does not require classification for skin irritation or corrosion according to Regulation (EC) 1272/2008.

Eye Irritation

Regarding the potential for the substance to cause eye irritation, inconclusive results were obtained for the substance using the in vitro BCOP assay. Taking into account that the substance gave negative results in respective in vitro skin irritation and corrosion studies, and that the IVIS score obtained in the BCOP was 12.5 (e.g. at the lower end of the range of 3 to 55 indicated for “no prediction can be made”), in a precautionary approach, the substance has been classified in Category 2 for eye irritation according to Regulation (EC) 1272/2008. Further investigations of the eye irritation potential of the substance are on-going.