N-Phenyl-diethanolamine, reaction products with formaldehyde

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 2020 to 23rd March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Storage conditions: At room temperature protected from light
Physical description: Light orange liquid

Test animals / tissue source

Species:

cattle

Strain:

other: n/a

Details on test animals or tissues and environmental conditions:

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and were tested the day of arrival in the laboratory.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 minutes ± 1 minutes

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and
neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 10 ± 1 minutes

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 ºC. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded.

POST-INCUBATION PERIOD: Yes. 120 ± 10 minutes at 32 ± 1°C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2 (MEM with phenol red; cMEM)
- POST-EXPOSURE INCUBATION: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to: Opacity = [(Io/I)-O.9894]/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490).
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Others (e.g, pertinent visual observations, histopathology): N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range, and UN GHS Category:
≤ 3, No Category
> 3; ≤ 55, No prediction can be made
>55, Category 1

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

6.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - the mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range
- Acceptance criteria met for positive control: Yes - the mean in vitro irritancy score was 42 and within two standards deviations of the current historical positive control mean

Any other information on results incl. tables

 Table 1
Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity ^1	Mean Permeability ^1	Mean In vitroIrritation Score ^1, 2
Negative control	2.6	0.019	2.9
Positive control (Ethanol)	19	1.521	42
Test item	6.4	1.134	23

¹     Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

²     In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀ value).

Table 2 Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity ^2	Mean Final Opacity
Negative control	1.6	3.9	2.2		2.6
	1.4	4.0	2.6
	2.2	5.2	3.0
Positive control	3.4	29.0	25.6	23	19
	3.6	25.3	21.8	19
	1.2	20.0	18.8	16
Test item	2.2	12.3	10.0	7.4	6.4
	2.4	11.4	9.0	6.4
	3.0	11.2	8.2	5.6

Calculations are made without rounding off.

1    Final Opacity = Opacity after treatment – Opacity before treatment.

²    Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

Table 3          
Permeability Score Individual Values (Uncorrected)

Treatment	Dilution factor	OD490	OD490	OD490	Average OD	Final OD	Mean final negative control
		1	2	3
Negative control	1	-0.004	-0.003	-0.002	-0.003	-0.003	0.019
	1	-0.004	-0.004	-0.002	-0.003	-0.003
	1	0.064	0.064	0.066	0.065	0.065
Positive control	1	1.395	1.355	1.389	1.380	1.380
	6	0.450	0.359	0.371	0.393	2.360
	1	0.974	0.991	0.972	0.979	0.979
Test item	1	1.138	1.110	1.122	1.123	1.123
	1	0.933	0.930	0.928	0.930	0.930
	1	1.442	1.411	1.365	1.406	1.406

Calculations are made without rounding off.

Table 4          
Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	1	1.376	1.336	1.370	1.360	1.360	1.521
	6	0.431	0.340	0.352	0.374	2.243
	1	0.955	0.972	0.953	0.960	0.960
Test item	1	1.119	1.091	1.103	1.104	1.104	1.134
	1	0.914	0.911	0.909	0.911	0.911
	1	1.423	1.392	1.346	1.387	1.387

Calculations are made without rounding off.

¹    OD₄₉₀ values corrected for the mean final negative control permeability (0.019).

 

Table 5          
In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy Score ^1
Negative control	2.2	-0.003	2.2
	2.6	-0.003	2.6
	3.0	0.065	4.0
Positive control	23	1.360	43
	19	2.243	53
	16	0.960	31
Test item	7.4	1.104	24
	6.4	0.911	20
	5.6	1.387	26

¹   In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀ value).

²   Positive control and test item are corrected for the negative control.

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the condition of this study, the test item induced an IVIS > 3 ≤ 55 and it was concluded that no prediction on the classification can be made in respect to its potential to cause eye irritation.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item, as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This study describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied undiluted (750 μL) directly on top of the corneas.

The mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 42 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item induced a mean in vitro irritancy score of 23 after 10 minutes of treatment which falls within the IVIS > 3 ≤ 55 category.

Under the condition of this study, the test item induced an IVIS > 3 ≤ 55 and it was concluded that no prediction on the classification can be made in respect to its potential to cause eye irritation.