Registration Dossier

Administrative data

Description of key information

A study was conducted on a structurally similar substance to the registered substance according to OECD 439 and GLP requirements to determine skin irritation potential. The substance was found to be non-irritating to the skin. A study was conducted on a structurally similar substance to the registered substance according to OECD 437 and GLP requirements. The substance was found to be classified as Category 1 (Irreversible effects on the eye) for severe eye damage, in accordance with EU CLP criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: In Vitro (Reconstructed Human Epidermis)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 439. The study was conducted on the substance 6-[(p-tosyl)amino]hexanoic acid, compound with 2,2’,2’’- nitrilotriethanol (1:1) (tosyl salt) which is structurally similar to the registered substance.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Reconstructed human epidermis
Strain:
other: Reconstructed human epidermis
Details on test animals and environmental conditions:
Receipt of Tissues

On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 17th June 2013). The maintenance medium was pre-warmed to 37°C. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 ml pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37°C ± 2°C in a humidified atmosphere of 5% CO2 in air.
Type of coverage:
other: The test substance was ground using a pestle and mortar and dispensed over each tissue using glass weighing boats.
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
The test substance was ground using a pestle and mortar. A weight of 10 mg ± 2 mg was dispensed over each tissue using glass weighing boats.
Duration of treatment / exposure:
15 minutes (triplicate tissues)
Observation period:
After exposure the tissues were incubated for 42 hours. After 42 hours the tissues were transferred to a well containing MTT and incubated for 3 hours.
Number of animals:
Not applicable
Details on study design:
The negative control was sterile Dulbecco's Phosphate Buffered Saline (DPBS) with magnesium and calcium. The positive control was 5% Sodium Dodecyl Sulphate (SDS) in purified water. The controls and their results were shared with another study performed in the same assay.

The test involves the application of the test substance for 15 minutes to the EPISKIN™ three-dimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38 cm2. The EPISKIN™ kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model (OECD 439) uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.
Irritation parameter:
other: Tissue viability
Basis:
mean
Score:
97.8
Max. score:
113.5
Reversibility:
no data
Remarks on result:
other: Mean of three replicate tissues
Other effects:
Negative control: The mean absorbance of the triplicate negative control values was 0.862 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 9.8 which was below the maximum value of 18.
Positive control: The percentage mean viability of the positive control was 25.7 ± 2.4 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

The mean Optical Density (OD) for the 6 replicate blanks was subtracted from the individual test substance and control tissues OD.

The viability of each tissue was expressed as a percentage of the mean negative control value.

Episkin Results

Sample

Tissue Replicate

Optical Density (OD)

OD – Blank

% Negative Control

Negative Control

a

1.033

0.994

0.891

0.852

101.1

b

0.916

0.913

0.775

0.771

89.7

c

1.117

1.049

0.975

0.907

109.2

Replicates a, b, c

Average sd

0.862

0.080

100.0

9.8

Positive Control

a

0.388

0.371

0.246

0.230

27.6

b

0.344

0.335

0.202

0.193

22.9

c

0.379

0.361

0.238

0.219

26.5

Replicates a, b, c

Average sd

0.221

0.021

25.7

2.4

Test Substance

a

1.139

1.100

0.998

0.958

113.5

b

0.944

0.907

0.803

0.765

91.0

c

0.931

0.886

0.789

0.744

88.9

Replicates a, b, c

Average sd

0.843

0.107

97.8

13.6

Blank

 

0.135

0.145

0.144

0.140

0.143

0.143

 

 

Average sd

0.142

0.004

 

sd: Standard Deviation

Note: Rounded values only are displayed; unrounded numbers are used for calculations by Excel spreadsheet

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It was concluded that the test substance had a mean tissue viability of 97.8% ± 13.6% and was predicted as non-irritant to the skin. The test substance is not classified as a skin irritant, in accordance with EU CLP criteria.
Executive summary:

The skin irritation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 439. It was concluded that the test substance had a mean tissue viability of 97.8% ± 13.6% and was predicted as non-irritant to the skin. The test substance is not classified as a skin irritant, in accordance with EU CLP criteria. Read-across between the registered substance and the test substance (tosyl salt) is considered justified owing to the structural similarity of the two substances. Both are salts of a carboxylic acid and triethanolamine. The acid components are structural isomers and differ only in the position of a single methyl group in the N-methyl salt. This is not expected to change the behavior or properties of the substances, and both substances have shown similarity in the results of other physical endpoints such as water solubility.

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 437. The study was conducted on the substance 6-[(p-tosyl)amino]hexanoic acid, compound with 2,2’,2’’- nitrilotriethanol (1:1) (tosyl salt) which is structurally similar to the registered substance.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Bovine Cornea
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Bovine corneas were supplied by a local abattoir. The eyes were removed after slaughter, 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. On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.
Vehicle:
other: sodium chloride
Controls:
yes
Amount / concentration applied:
The test article was tested as a 20% w/v suspension in 0.9% sodium chloride solution. A volume of 750 µl of the test article formulation was applied to each of three corneas followed by a four hour incubation at 32°C.
Duration of treatment / exposure:
Four hour incubation
Observation period (in vivo):
After the incubation period the cornea were washed once in media without phenol red and incubated for a further 1.5 hours ± 5 minutes
Number of animals or in vitro replicates:
Three bovine corneas were tested.
Details on study design:
Fresh corneas, mounted onto specifically designed holders were treated topically with the test material. Eye corrosion/severe irritation potential was based on the combined effect of the test substance on the opacity of the cornea following the treatment and the cornea’s ability to resist penetration of a fluorescent dye through the tissue.
Irritation parameter:
other: corneal opacity
Basis:
mean
Time point:
other: 4 hour incubation after application of test substance
Score:
30
Max. score:
34.7
Reversibility:
not specified
Remarks on result:
other: score for test substance
Irritation parameter:
other: corneal opacity
Basis:
mean
Time point:
other: 4 hour incubation after application of negative control
Score:
0
Max. score:
1.7
Reversibility:
not specified
Remarks on result:
other: score for negative control
Irritation parameter:
other: corneal opacity
Basis:
mean
Time point:
other: 4 hour incubation after application of positive control
Score:
60
Max. score:
69.7
Reversibility:
not specified
Remarks on result:
other: score for positive control
Irritation parameter:
other: corneal permeability
Basis:
mean
Time point:
other: Total incubation of 5.5 hours after application of the test substance
Score:
2.031
Max. score:
2.037
Reversibility:
not specified
Remarks on result:
other: score for test substance
Irritation parameter:
other: corneal permeability
Basis:
mean
Time point:
other: Total incubation of 5.5 hours after application of negative control
Score:
0
Max. score:
0.129
Reversibility:
not specified
Remarks on result:
other: score for negative control
Irritation parameter:
other: corneal permeability
Basis:
mean
Time point:
other: Total incubation of 5.5 hours after application of positive control
Score:
1.78
Max. score:
1.986
Reversibility:
not specified
Remarks on result:
other: score for positive control
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: Total incubation of 5.5 hours
Score:
60.47
Reversibility:
not specified
Remarks on result:
other: score for test substance
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: Total incubation of 5.5 hours
Score:
0
Reversibility:
not specified
Remarks on result:
other: score for negative control
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: Total incubation of 5.5 hours
Score:
86.7
Reversibility:
not specified
Remarks on result:
other: score for positive control
Irritant / corrosive response data:
The test substance produced an In Vitro Irritation Score of 60.47.

Corneal Opacity

Substance

Cornea number

Initial opacity

Post incubation opacity

Change in opacity

Mean change in opacity

Corrected opacity

Mean corrected opacity

Test substance

15

1

36

35

N/A

33.7

30

16

1

37

36

34.7

35

1

24

23

21.7

Negative control

3

0

3

3

1.33

1.7

0

9

1

2

1

-0.3

10

0

0

0

-1.3

Positive control

19

0

58

58

N/A

56.7

60

20

0

71

71

69.7

21

0

55

55

53.7

  

Corneal Permeability

Substance

Cornea number

Mean blank OD490

OD490

Corrected OD490

Mean corrected OD490

Final corrected OD490

Mean group corrected OD490

Test substance

15

-

2.128

2.128

N/A

2.030

2.031

16

2.136

2.136

2.037

35

2.126

2.126

2.027

Negative control

3

0

0.035

0.035

0.099

-0.063

0

9

0.228

0.228

0.129

10

0.033

0.033

-0.066

Positive control

19

-

2.085

2.085

N/A

1.986

1.780

20

1.794

1.794

1.695

21

1.758

1.758

1.660

 

OD490 = Optical Density at 490 nm

Calculated In Vitro Irritation Score (IVIS)

Substance

Mean opacity

Mean permeability

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

Negative control

0

0

0

Positive control

60

1.78

86.7

Test substance

30

2.031

60.47

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance produced an In Vitro Irritation Score (IVIS) of 60.47 and was considered to be corrosive or severely irritating to the eye. The test substance is classified in Category 1 (Irreversible effects on the eye) for severe eye damage, in accordance with EU CLP criteria.
Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 437. The test substance was applied (in vitro) to three bovine corneas, with a positive and a negative control applied to further groups of three bovine cornea. Corneal opacity was recorded after a 4 hour incubation period. The cornea were washed after the initial 4 hour incubation and incubated for a further 1.5 hours before corneal permeability was determined. The mean corneal opacity and mean corneal permeability scores were used to calculate a mean In Vitro Irritation Score (IVIS). The test substance produced an IVIS of 60.47 and was considered to be corrosive or severely irritating to the eye (an IVIS >55.1 indicates a corrosive or severe irritant to eyes). All three cornea exposed to the test material had a corneal opacity score of >20, with an average score of 30. It is not expected that the effects to the cornea would be fully reversible, the test substance is therefore classified in Category 1 (Irreversible effects on the eye) for severe eye damage, in accordance with EU CLP criteria. Read-across between the registered substance and the test substance (tosyl salt) is considered justified owing to the structural similarity of the two substances. Both are salts of a carboxylic acid and triethanolamine. The acid components are structural isomers and differ only in the position of a single methyl group in the N-methyl salt. This is not expected to change the behavior or properties of the substances, and both substances have shown similarity in the results of other physical endpoints such as water solubility.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The skin irritation potential of the registered substance was determined via read-across to the results of testing on the structurally similar substance tosyl salt. The tosyl salt was tested in accordance with the OECD Guideline for Testing of Chemicals 439. It was concluded that the test substance had a mean tissue viability of 97.8% ± 13.6% and was predicted as non-irritant to the skin. The test substance is not classified as a skin irritant, in accordance with EU CLP criteria.

The eye irritation potential of the registered substance was determined via read-across to the results of testing on the structurally similar substance tosyl salt. The tosyl salt was tested in accordance with the OECD Guideline for Testing of Chemicals 437. The test substance was applied (in vitro) to three bovine corneas, with a positive and a negative control applied to further groups of three bovine cornea. Corneal opacity was recorded after a 4 hour incubation period. The cornea were washed after the initial 4 hour incubation and incubated for a further 1.5 hours before corneal permeability was determined. The mean corneal opacity and mean corneal permeability scores were used to calculate a mean In Vitro Irritation Score (IVIS). The test substance produced an IVIS of 60.47 and was considered to be corrosive or severely irritating to the eye (an IVIS >55.1 indicates a corrosive or severe irritant to eyes). All three cornea exposed to the test material had a corneal opacity score of >20, with an average score of 30. It is not expected that the effects to the cornea would be fully reversible, the test substance is therefore classified in Category 1 (Irreversible effects on the eye) for severe eye damage, in accordance with EU CLP criteria.

Read-across between the registered substance and the test substance (tosyl salt) is considered justified owing to the structural similarity of the two substances. Both are salts of a carboxylic acid and triethanolamine. The acid components are structural isomers and differ only in the position of a single methyl group in the N-methyl salt. This is not expected to change the behavior or properties of the substances, and both substances have shown similarity in the results of other physical endpoints such as water solubility.


Justification for selection of skin irritation / corrosion endpoint:
Study was conducted on a structurally similar substance to the registered substance according to OECD 439 and GLP requirements. The substance was found to be non-irritating to the skin.

Justification for selection of eye irritation endpoint:
Study was conducted on a structurally similar substance to the registered substance according to OECD 437 and GLP requirements. The substance was found to be classified as Category 1 (Irreversible effects on the eye) for severe eye damage, in accordance with EU CLP criteria.

Effects on eye irritation: highly irritating

Justification for classification or non-classification

The skin irritation study demonstrated that the substance was not expected to be a skin irritant and did not require classification in accordance with EU CLP criteria. The eye irritation study demonstrated that the substance had the potential to cause severe, irreversible eye damage and therefore the substance requires classification as a Category 1 Eye Irritant in accordance with EU CLP criteria.