1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Sep 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-3,4-dihydroquinolin-l (2H)-yl)ethanone

EC Number:

855-228-0

Cas Number:

4497-59-0

Molecular formula:

C14 H19 N O

IUPAC Name:

1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

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 (e.g. age, sex, weight): 14 month; no further characteristics given in the report
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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 corneas 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 corneas 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:

physiological saline

Remarks:

20% suspension (w/v) in saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20% in saline, using sonication for 10 minutes

VEHICLE
- Amount(s) applied (volume or weight unit): 0.75 mL
- Concentration (if solution): 0.9% saline

Duration of treatment / exposure:

240 min

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

90 ± 5 min at 32 ± 1 °C (permeability measurement)

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 each 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 240 minutes.

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 MEM (comparable to 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 90 ± 5 min (permeability measurement).

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 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 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.
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 corneas

Run / experiment:

240 min

Value:

1.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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 ( 0.064 ± 0.006) 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 (89.47 ± 1.74) which was within two standard deviations of the current historical mean (see 'any other information on results incl. tables', tables 3 and 4 (historical controls)).

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 (t240-t0) [Opacity Units]	Corrected Opacity
		After subtraction of the background opacity	Mean of group	Standard deviation
Negative control	1		0.33	0.58
	0
	0
Test substance	1	0.67	0.33	0.58
	0	-0.33*
	1	0.67
Positive control	84	83.67	84.67	1.73
	89	86.67
	84	83.67

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

 

 

Table 2: Permeability [OD] values 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	0.064		0.064	0.006
	0.059
	0.070
Test substance	0.134	0.070	0.083	0.044
	0.196	0.132
	0.112	0.048
Positive control	0.390	0.326	0.32	0.01
	0.385	0.321
	0.378	0.314

 

 

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

	Per Cornea	Per Group
		Mean	SD
Negative control	1.96	1.30	0.58
	0.89
	1.05
Test substance	1.71	1.69	0.30
	1.98
	1.38
Positive control	88.55	89.47	1.74
	91.48
	88.37

 

 

Table 4: Historical Control Values of the BCOP assay with solid test items.

	Positive control	Negative control
Mean IVIS	112.81	1.29
Standard Deviation of IVIS	12.88	0.32
Range of IVIS	87.39-146.55	0.77-2.85
95% Control limits of IVISpos	87.04-138.57
Mean Opacity t240min	109.50	0.25
Standard deviation of Opacity t240min	54.00-187.00	0.00-1.33
Range of Opacity t240min	19.06	0.32
Mean Permeability	0.22	0.07
Standard Deviation of Permeability	0.29	0.01
Range of Permeability	0.00-1.96	0.05-0.10

Values of 87 studies with solid test items sharing 49 sets of controls, performed between Jan 2016 – Feb 2020.

 

Applicant's summary and conclusion

Interpretation of results:

other: Eye Irrit. 2 (H319) according to Regulation (EC) No 1272/2008

Conclusions:

This study was part of an integrated testing strategy. A bottom-up approach starting with the Epiocular™ test (according to OECD 492) was conducted. A positive in vitro irritation response was revealed for the test substance, which is not conclusive with respect to classification of the test substance as eye irritant (Eye Irritant Cat. 2) or serious eye damage (Eye Damage Cat. 1). Therefore, the BCOP was chosen as follow-up test (according to OECD 437) and, as already described, the test substance turned out not to be severe irritating/corrosive.

It is therefore concluded based on OECD 492 that the test substance 1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone should be classified as Eye Irrit. Cat. 2 according to Regulation (EC) No 1272/2008.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of 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 20% (w/v) suspension

in saline (0.9% (w/v) NaCl in deionised water) of the test item [trade name] as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 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 and opacity was measured again (t240).

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 (physiological saline), neither an increase of opacity nor permeability of the corneae was observed.

The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/GHS Category 1).

Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.69. According to OECD 437 (see table in chapter 3.9.3) the test item is not categorized (EU CLP/GHS No Category).