2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

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)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate

EC Number:

221-309-9

EC Name:

2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate

Cas Number:

3063-94-3

Molecular formula:

C7H6F6O2

IUPAC Name:

2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 449935-248
- Expiration date of the lot/batch: 30 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refridgerator (2-8ºC)

Test animals / tissue source

Species:

other: Bovine eyes

Strain:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

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.
Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µl
- Concentration (if solution): as supplied

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

3

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 BASF (Ludwigshafen, 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 ± 1ºC. The corneas were incubated for the minimum of 1 hour at 32 ± 1ºC.

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, BASF, Ludwigshafen, Germany). 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.
QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
Ethanol, Identification number RS532, Batch number K47177483

APPLICATION DOSE AND EXPOSURE TIME
750µl of either the negative control, positive control (Ethanol) or test item. 10 minute exposure time

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- POST-EXPOSURE INCUBATION: 120 ± 10 minutes

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=(I_0/I-0.9894)/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)

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 UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

DECISION CRITERIA: The assay is considered acceptable if:
• The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
• The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean opacity score

Value:

-0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

mean in vitro irritation score

Value:

-0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Results

2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate was tested neat. 

Table1 summarizes the opacity, permeability andin vitroirritancy scores of the test item and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2- 5. 

The individual in vitro irritancy scores for the negative controls ranged from 0.3 to 2.3. The individual positive control in vitro irritancy scores ranged from 37 to 53. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from -0.8 to -0.4 and permeability values ranging from 0.001 to 0.003. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.8 to -0.3 after 10 minutes of treatment with 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate.

Table 1: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In Vitro Irritation Score^1,2
Negative control	1.6	-0.004	1.5
Positive control (ethanol)	21	1.751	48
Test Item	-0.5	0.002	-0.5

1 -Calculated using the negative control 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 Scores

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	1.0	1.5	0.4		1.6
	3.2	5.1	2.0
	4.9	7.3	2.4
Positive control	1.4	21.4	20.1	18	21
	1.7	26.5	24.8	23
	1.7	26.1	24.4	23
Test Item	2.4	3.5	1.1	-0.5	-0.5
	4.3	5.1	0.8	-0.8
	2.7	3.9	1.2	-0.4

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

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

³    Calculations are made without rounding off.

Table 3: Permeability Score individual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	-0.010	-0.010	-0.008	-0.009	-0.009	-0.004
	1	-0.003	-0.004	-0.004	-0.001	-0.001
	1	-0.003	-0.003	-0.002	-0.003	-0.003
Positive control	1	1.219	1.235	1.234	1.229	1.229
	6	0.325	0.331	0.333	0.0330	1.978
	6	0.326	0.327	0.342	0.332	1.990
Test Item	1	-0.005	-0.006	0.001	-0.003	-0.003
	1	-0.001	-0.002	-0.004	-0.002	-0.002
	1	-0.002	-0.003	0.002	-0.001	-0.001

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	1.223	1.239	1.238	1.234	1.234	1.751
	6	0.329	0.335	0.337	0.334	2.004
	6	0.330	0.331	0.346	0.336	2.016
Test Item	1	-0.001	-0.002	0.005	0.001	0.001	0.002
	1	0.003	0.002	0.000	0.002	0.002
	1	0.002	0.001	0.006	0.003	0.003

¹    OD₄₉₀ values corrected for the mean final negative control permeability (-0.004).

²    Calculations are made without rounding off.

Table 5: In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy Score^1
Negative control	0.4	-0.009	0.3
	2.0	-0.001	1.9
	2.4	-0.003	2.3
Positive control	18	1.234	37
	23	2.004	53
	23	2.016	53
Test Item	-0.5	0.001	-0.5
	-0.8	0.002	-0.8
	-0.4	0.003	-0.3

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:

GHS criteria not met

Conclusions:

Since 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate 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 report 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 study procedures described in this report were based on the most recent OECD guideline.

Batch 449935-248 of the test item was a clear colourless liquid. The test item was applied as it is (750 µl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 48 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 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 10 minutes of treatment. 

In conclusion, since 2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.