Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl]-2-oxoethyl]-4-methyl-, chloride (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.02.2011-08.09.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

EC Number:

700-714-9

Cas Number:

1254469-57-2

Molecular formula:

C14H20ClN3O3

IUPAC Name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

Details on test material:

- Name of test material (as cited in study report): Tinocat ES 96000
- Physical state: Solid, white
- Analytical purity: 98.7 g/100 g (see analytical report, study code 11L00011)
- Lot/batch No.: RD173323
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed
- Storage condition of test material: Room temperature (protected from light, protected against humidity)

Test animals / tissue source

Species:

other: bovine cornea

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

water

Remarks:

highly de-ionized

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20% (w/v)

VEHICLE

- Purity: highly de-ionized water

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

Corneas free of defects were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders, filled to excess with prewarmed Eagles’s MEM and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 566 opacity units1 were discarded. Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the 20% (w/v) test-substance preparation was applied into the anterior chamber using a pipette. Control tissues were concurrently applied into the anterior chamber with 750 μL of highly deionized
water (negative control, NC) or with 750 μL of 20% (w/v) solution of Imidazole in highly de-ionized water (positive control, PC). The corneas were incubated at about 32 °C for approximately 4 hours. The test substance, NC and PC were then removed and the epithelium was washed at least 3 times with Eagle’s MEM. Both chambers were then refilled with fresh Eagle’s MEM.
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In Vitro Irritancy Score IVIS, control

Basis:

mean

Remarks:

3 corneas

Time point:

other: 4h

Score:

5.1

Remarks on result:

other: mean corneal opacity and permeability values were used to calculate an In Vitro Irritancy Score (IVIS)

Results 2

Irritation parameter:

other: In Vitro Irritancy Score (IVIS), test item

Basis:

mean

Remarks:

3 corneas

Time point:

other: 4h

Score:

14.5

Remarks on result:

other: IVIS > 55 risk of serious damage to the eyes; IVIS ≤ 55 no risk of serious damage to the eyes, IVIS (positive control) = 105.7

Any other information on results incl. tables

Opacity score

Test substance	Mean	SD
11/0013-1	11.3	8.7
negative control	4.5	0.8
positive control	64.3	7.9

Permeability score

Test substance	Mean	SD
11/0013-1	0.215	0.148
negative control	0.040	0.051
positive control	2.757	0.605

In vitro irritancy score (IVIS)

Test substance	IVIS	SD
11/0013-1	14.5	9.6
negative control	5.1	1.2
positive control	105.7	10.5

An in vitro irritancy score of greater than 55 is an indication for risk of serious damage to the eyes.

Calculation of the corneal opacity value

First, the opacity was calculated using the opacitometer specific algorithm, with a and b device specific and with Io being the illuminance (lux) through the empty corneal holder with windows and liquid and I being the illuminance (lux) through the holder with the cornea (more details see specific SOP). • opacity value = a * Io/I + b Then the opacity change per cornea was calculated by subtracting the initial from the final opacity. • opacity change per cornea = final opacity - initial opacity Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control. • corrected opacity change = opacity change - mean opacity change of NC

Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group. • mean opacity value = mean of all corrected opacity changes per group 3.8.2. Calculation of permeability value First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea. • OD490 value = OD490 - mean blank OD490 If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value: • OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490) Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control. • corrected OD490 value = OD490 value - mean OD490 value of NC Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group. • mean OD490 value = mean of all corrected OD490 values per group 3.8.3.

Calculation of the In Vitro Irritancy Score (IVIS) The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined: • IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change • IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

Applicant's summary and conclusion

Interpretation of results:

other: no irreversible effects on the eye

Conclusions:

Based on the observed results and applying the evaluation criteria cited in chapter 3.10 it was concluded, that Tinocat ES 96000 does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen. The test method does not yet allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at
present, results from another study would be needed.

Executive summary:

The potential of Tinocat ES 96000 to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas. Based on the observed results and applying the evaluation criteria it was concluded, that Tinocat ES 96000 does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test). The test method does not yet allow for the evaluation of eye irritation.