2,4,6-trimethylpyridine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

9th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trimethylpyridine

EC Number:

203-613-3

EC Name:

2,4,6-trimethylpyridine

Cas Number:

108-75-8

Molecular formula:

C8H11N

IUPAC Name:

2,4,6-trimethylpyridine

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Storage, temperature and transport conditions of ocular tissue:
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Selection and preparation of corneas:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement. At the end of the incubation period, the basal opacity was determined. Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min +/- 30 sec

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 10 min exposure


POST-INCUBATION PERIOD:
yes, 2 h

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability:
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was measured with a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The mean IVIS value of each treated group was calculated from the individual IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS ≤ 3: UN GHS No Category
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55: UN GHS Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

10 min treatment time, mean of three corneas

Value:

52.43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.87).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 98.87) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
The test item 2,4,6-Collidine was tested undiluted. Relative to the negative control, the test item 2,4,6-Collidine caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 52.43 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Any other information on results incl. tables

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation in vitro score	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0	0.059	0.058	0.89	0.87	0.03	No Category
	0		0.059		0.89
	0		0.059		0.83
Positive Control	82*		0.843*		94.65	98.87	4.26	Category 1
	80*		1.252*		98.79
	87*		1.078*		103.18
2,4,6-Collidine	20*		2.201*		53.02	52.43	1.88	No prediction can be made
	23*		2.063*		53.95
	18*		2.155*		50.33

*corrected values (the mean permeability OD of the negative control is subtracted from the permeability OD of each treated cornea)

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made based on the calculated IVIS score of 52.43.

Executive summary:

The in vitro study according to OECD Guideline 437 was performed to assess the corneal damage potential of 2,4,6-Collidine by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water), neither an increase of opacity nor permeability of the corneae was observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/UN GHS (Cat 1)). Relative to the negative control, the test item 2,4,6-Collidine caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 52.43 according to OECD 437 (see table in chapter 3.8.3).

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (UN GHS) for 2,4,6-Collidine.