N,N'-methylenedistearamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jul 2018 to 24 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-methylenedistearamide

EC Number:

203-657-3

EC Name:

N,N'-methylenedistearamide

Cas Number:

109-23-9

Molecular formula:

C37H74N2O2

IUPAC Name:

N,N'-methylenedistearamide

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Characteristics of donor animals: typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics. They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: within one day
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection and after incubation in Bovine Corneal Opacity and Permeability (BCOP) holders. Only corneas free of damage were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
For the purpose of this study the test item was used as supplied as the test item could not be formulated to a concentration of 20% w/v in sodium chloride 0.9% w/v as a suitable suspension/solution could not be obtained. An appropriately sized disc of the test item was applied to entirely cover the cornea and was found to weigh approximately 0.4900g.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

A post-treatment opacity reading was performed immediately after removal of the test substance or controls. Followed an incubation time of 90 minutes for the permeability assay, as is described in details on study design.

Number of animals or in vitro replicates:

Triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 85 minutes. At the end of the incubation period each cornea was examined for defects.

QUALITY CHECK OF THE ISOLATED CORNEAS
Only corneas free of damage were used. The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken as reference point for each cornea, using a calibrated opacitometer.

NUMBER OF REPLICATES
Triplicates

NEGATIVE CONTROL USED
The negative control item, sodium chloride 0.9% w/v, was used as supplied.

POSITIVE CONTROL USED
The positive control item, Imidazole (purity >99%), was used as a 20% w/v solution in sodium chloride 0.9% w/v.

APPLICATION DOSE AND EXPOSURE TIME
For the purpose of this study the test item was used as supplied as the test item could not be formulated to a concentration of 20% w/v in sodium chloride 0.9% w/v as a suitable suspension/solution could not be obtained. An appropriately sized disc of the test item was applied to entirely cover the cornea and was found to weigh approximately 0.4900g.

TREATMENT METHOD: [closed chamber / open chamber]
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.4900 g of the solid test item was found to adequately cover the corneal surface. 0.75mL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. However, no histopathology was performed.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
The in vitro irritancy scores are summarized in Table 1 in 'Any other information on material and methods incl. tables'.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 71.2 to 132.9.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.044.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Based on #1, #2, #3

Value:

0.3

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Remarks:

corrected value

Run / experiment:

Mean #1, #2, #3

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein leakage

Remarks:

corrected value

Run / experiment:

Mean #1, #2, #3

Value:

0.023

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Individual and mean corneal opacity and permeability measurements can be found in Table 1 in 'Any other information on results incl. tables'.

Any other information on results incl. tables

 Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	6	9	3		0.006
	2	4	4	0		0.000
	3	4	7	3		0.002
				2.0*		0.003##		2.0
Positive Control	4	5	72	67	65.0	1.075	1.072
	5	4	67	63	61.0	2.115	2.112
	6	3	62	59	57.0	1.430	1.427
					61.0#		1.537###	84.1
Test Item	7	6	8	2	0.0	0.051	0.048
	9	5	4	-1	0.0	0.011	0.008
	10	6	5	-1	0.0	0.014	0.011
					0.0#		0.023###	0.3

OD = Optical density

* Mean of the post-treatment

# Pre-treatment corrected values

## Mean permeability

## Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

This Bovine Corneal Opacity and Permeability test (BCOP), performed according to OECD 437 under GLP, indicated that the test item is not considered to be irritating to the eye.

Executive summary:

In this this eye irritation study performed according to OECD 437 under GLP, the irritative potential of the test substance was assessed ex vivo in a Bovine Corneal Opacity and Permeability test (BCOP). Bovine eyes were obtained from a local abattoir and the corneas were prepared and mounted in BCOP holders. Only corneas free of damage were used. Approximately 0.4900 g of the solid test item was found to adequately cover the corneal surface. For the negative control item (sodium chloride 0.9% w/v), and positive control item, (Imidazole (purity >99%)), 0.75 mL was applied to the corneas. The test was performed in triplicates. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. After exposure, the corneas were washed 3 times with EMEM with phenol red, before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated.

The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment. The in vitro irritancy scores are summarized as follows: test item 0.3, negative control 2.0, positive control 84.1. The controls were within the acceptable range, indicating the validity of the study. In conclusion, the test substance was not considered to be irritating to the eye.