1-(2,2,4-trimethyl-1-quinolyl)ethanone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Oct 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:

adopted 26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

EC Number:

665-911-3

Cas Number:

5855-23-2

Molecular formula:

C14 H17 N O

IUPAC Name:

1-(2,2,4-trimethyl-1,2-dihydroquinolin-1-yl)ethan-1-one

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained from slaughterhouse (AB Schlachthof GmbH & Co. KG, Aschaffenburg, Germany)
- Characteristics of donor animals: 14 month old; no further characteristics given in the report
- Storage, temperature and transport conditions of ocular tissue: stored in Hanks´ Balanced Salt Solution (HBSS) in the cooled slaughterhouse 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.
- Time interval prior to initiating testing: same day
- Indication of any existing defects or lesions in ocular tissue samples: no; only corneae from eyes free of defects were used
- Indication of any antibiotics used: yes, 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin)

SELECTION AND PREPARATION OF CORNEAS
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period, the medium was changed before basal opacity measurement (t0). Only corneae with a value of the basal opacity < 7 were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL (also for neg. and pos. controls)

Duration of treatment / exposure:

10 min ± 30 sec at 32 ± 1 °C

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

single experiment with triplicates for each treatment and control group

Details on study design:

TREATMENT METHOD: Open-Chamber method: 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. The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted 10 minutes (± 30 seconds).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3. After exposure, the test item or control items, respectively, were rinsed off from the application side with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium is free of test item the corneas are given a final rinse with cMEM (MEM supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum) without phenol red.

POST-EXPOSURE INCUBATION:
The corneas were incubated at 32 ± 1 °C for two hours in a vertical position.

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, 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 corneae.
- 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 min 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:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP and GHS-UN, respectively.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneae

Run / experiment:

10 min exposure

Value:

4.61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made (according to OECD TG 437)

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control resulted in opacity (0.33 ± 0.58) and permeability (0.060 ± 0.010) values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control (see 'any other information on results incl. tables', table 1 and 2, respectively).
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS (116.22 ± 11.30) which was within two standard deviations of the current historical mean (see 'any other information on results incl. tables', table 3).

HISTORICAL CONTROL DATA
See 'any other information on results incl. tables', table 4

Any other information on results incl. tables

Table 1: Opacity values and corrected opacity values of the BCOP assay.

Parameter	Opacity (t130-t0) [Opacity Units]	Corrected Opacity
		After subtraction of the background opacity	Mean of group	Standard deviation
Negative control	0	-	0.33	0.58
	1
	0
Test substance	3	2.67	4.33	2.08
	4	3.67
	7	6.67
Positive control	79	78.67	92.00	12.58
	104	103.67
	94	93.67

 

 

Table 2: Permeability [OD] values at 490 nm of the BCOP assay

Parameter	Permeability at 490 nm [OD]	Corrected Permeability [OD] After subtraction of the background permeability	Mean of Triplicates	Standard deviation
Negative control	0.069		0.060	0.010
	0.060
	0.050
Test substance	0.114	0.054	0.014	0.035
	0.056	-0.004*
	0.052	-0.008*
Positive control	1.770	1.710	1.61	0.09
	1.603	1.543
	1.650	1.590

*Negative values of corrected permeability are set to zero for further calculation

 

Table 3: In-Vitro Irritancy Score (IVIS) values of the BCOP assay

	Per Cornea	Per Group
		Mean	SD
Negative control	1.04	1.23	0.60
	1.90
	0.75
Test substance	3.48	4.61	1.79
	3.67
	6.67
Positive control	104.32	116.22	11.30
	126.82
	117.52

 

Table 4: Historical Control Values of the BCOP assay with liquid test items (January 2016 - September 2020 (n = 101))

	Positive control	Negative control
Mean IVIS	94.58	1.02
Standard Deviation of IVIS	13.51	0.41
Range of IVIS	65.57 - 130.79	0.00 - 2.17
95% Control limits of IVISpos	68.00 - 122.00
Mean Opacity t130 min	77.49	0.10
Standard deviation of Opacity t130 min	14.21	0.41
Range of Opacity t130 min	18.67 - 124.67	0.00 - 0.67
Mean Permeability	1.14	0.06
Standard Deviation of Permeability	0.39	0.01
Range of Permeability	0.19 - 2.07	0.05 - 0.10

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Eye Corrosive Cat. 1) based on a positive result in the BCOP test method. A result for which no prediction can be made is not conclusive with respect to non-classification or classification as an eye irritant and therefore requires further evaluation and/or data generation. Under the present test conditions 1-(2,2,4-trimethylquinolin-l (2H)-yl)ethanone is non-corrosive to fresh bovine corneae in the Bovine corneal opacity and permeability (BCOP) test method (according to OECD 437). No prediction can be made with respect to non-classification or classification as an eye irritant.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of the test item [trade name] 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/GHS (Cat 1)).

Relative to the negative control, the test item caused an increase of the corneal opacity. The calculated mean in vitro irritancy score was 4.61.

According to OECD 437 (see table in chapter 3.9.3) a prediction for corneal irritation and damage potential cannot be made.