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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - November 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate
EC Number:
EC Name:
2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate
Cas Number:
Molecular formula:
2,2,2-trifluoro-1-(trifluoromethyl)ethyl methacrylate
Test material form:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: 449935-248
- Expiration date of the lot/batch: 30 March 2018

- Storage condition of test material: In refridgerator (2-8ºC)

Test animals / tissue source

other: Bovine eyes
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

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- 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:
Details on study design:
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.


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.

Ethanol, Identification number RS532, Batch number K47177483

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

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

- 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:
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

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean opacity score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Irritation parameter:
in vitro irritation score
Run / experiment:
mean in vitro irritation score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables


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 Opacity1   Mean Permeability1  Mean In Vitro Irritation Score1,2
 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 Opacity1   Negative control corrected Final Opacity2  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 -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 1.219  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 -0.005  -0.006  0.001  -0.003  -0.003   
   1 -0.001  -0.002  -0.004  -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 11    Negative control corrected OD490 21   Negative control corrected OD490 31  Negative control corrected OD490 Average Negative control corrected final OD490  Average OD 
 Positive control 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 -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  Final OD4902  In vitro Irritancy Score1
 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
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.