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

Endpoint summary

Description of key information

A study was conducted  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 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:

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 439.

Qualifier:

according to guideline

Guideline:

other: OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Reconstructed human epidermis

Strain:

other: Reconstructed human epidermis

Details on test animals or test system 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.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

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)

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.

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 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 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.

Justification for selection of skin irritation / corrosion endpoint:

Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 439.

Justification for selection of eye irritation endpoint:

Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 439.

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.