Registration Dossier

Toxicological information

Eye irritation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Waxy solid
Details on test material:
Appearance: Orange/brown waxy solid
Identity: 6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
Storage conditions: 15 to 30°C in a sealed container, protected from the light
Purity: 98%

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vivo

Resultsopen allclose all
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.