Reaction mass of 1,5-naphthylene diisocyanate and Amines, hydrogenated tallow alkyl

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1st February 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)

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Molecular formula:

C24H32N4O2

IUPAC Name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Obtained from a local abattoir
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 month old adult cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were transported on the day of slaughter and prepared immediately on arrival in the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: After being excised corneas were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (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
- Amount(s) applied (volume or weight with unit): Approximately 0.3270 g of the solid test item was found to adequately cover the corneal surface.

VEHICLE: not applicable

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. Additionally, a pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer and the average opacity for all corneas was calculated.

NUMBER OF REPLICATES
3 replicate corneas were used for the test item, positive and negative control treatments.

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v solution

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.

APPLICATION DOSE AND EXPOSURE TIME
Approximately 0.4915 g of the solid test item or 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.

TREATMENT METHOD: [closed chamber / open chamber]
Open chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION: 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability:Ppassage of sodium fluorescein dye measured at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader
- Others (e.g, pertinent visual observations, histopathology): Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled 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 required for this study

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria using the following prediction models:
- IVIS = ≤ 3 No category for UN GHS criteria and not classified under EU CLP
- IVIS = > 3 and ≤ 55 No prediction can be made for UN GHS and EU CLP criteria
- IVIS = > 55 Category 1 UN GHS and EU CLP criteria

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean Test item

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive and negative control acceptability criteria were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: Not reported

Any other information on results incl. tables

Table 2: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment - Pre-Treatment	Corrected Value	Uncorrected	Corrected
Negative Control	1	3	5	2		0.014
	2	2	6	4		0.024
	3	2	3	1		0.017
	Mean			2.3*		0.018**		2.6
Positive Control	6	5	93	88	85.7	0.878	0.860
	7	1	68	67	64.7	1.108	1.090
	9	6	80	74	71.7	2.825	2.807
	Mean				74.0***		1.585***	97.8
Test Item^1	10	2	3	1	0.0	0.080	0.062
	11	2	3	1	0.0	0.018	0.000
	12	2	3	1	0.0	0.011	0.000
	Mean				0.0***		0.021***	0.3

OD = Optical Density; * = Mean of post-treatment - pre-treatment; ** = Mean permeability; *** = Mean corrected values; ¹ The test item could not be formulated to a concentration of 20 % w/v solution in sodium chloride 0.9 % w/v

Table 3: Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control	1	Clear
	2	Clear
	3	Clear
Positive Control	6	Cloudy
	7	Cloudy
	9	Cloudy
Test Item	10	Clear
	11	Clear
	12	Clear

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean in vitro irritancy score for the item is 0.3, and based on the assessment criteria the test item is not classified under the EU CLP regulations and is assigned no category under the UN GHS criteria.

Executive summary:

The eye irritation potential of the test item was assessed in a OECD 437 and EU B.47 method guideline study. Approximately 0.3270 g of the test item was applied to the corneal surface of prepared adult cattle cornea, obtained on the day of extraction, and incubated for 240 minutes followed by rinsing to remove the test item. Additionally, sodium chloride (0.9 % w/v) and Imidazole (20 % w/v solution in 0.9 % w/v sodium chloride) were tested as negative and positive controls alongside the test item analysis. The opacity of treated corneas was assessed using a opacitometer and the permeability was determined at OD492 by microplate reader. The In vitro irritancy score (IVIS), based on the mean opacity and permeability measurement, was calculated for the test item, positive and negative controls; and the classification requirement was assessed based on the IVIS.

 

The mean IVIS for the item is 0.3, and based on the assessment criteria the test item is not classified under the EU CLP regulations and is assigned no category under the UN GHS criteria; though it is noted that the test item could not be formulated to a concentration of 20% w/v solution in sodium chloride 0.9% w/v. This did not have an effect on the study results. The mean IVIS for the negative and positive controls were 2.6 and 97.8, respectively, and therefore the acceptability criteria of the study were met.

 

The study is a GLP compliant guideline experimental study, and is deemed acceptable without restrictions for assessment of this endpoint.