6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 437.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tosyl salt

IUPAC Name:

Tosyl salt

Constituent 2

Reference substance name:

6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

EC Number:

301-097-5

EC Name:

6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

Cas Number:

93981-14-7

Molecular formula:

C13H19NO4S.C6H15NO3

IUPAC Name:

2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium 6-{[(4-methylphenyl)sulfonyl]amino}hexanoate

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

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Results 7

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

Results 8

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

Results 9

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.