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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 4 September 2017 to 4 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
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)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Kurimate
- CAS number: 1864727-96-7
- Analytical purity: 99.4%
- Physical state: Clear colorless liquid
- Lot number: S17-6-100
- Expiration date: One year from the shipping date (7 August 2017)
- Storage condition of test material: room temperature, air in the headspace of the container was replaced with nitrogen gas, in a dark place

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
BOVINE EYES
- Source: Fifteen eyes from cattle (26 to 29 months old) excised as soon as possible after slaughter at a slaughterhouse (Kumamoto Chuou Meat Center Co.,Ltd., 548, Subayashi, Toyono-machi, Uki-shi, Kumamoto 861-4307, Japan) were transported in a cooler with Hanks’ Balanced Salt Solution to the test facility. The bovine eyes were used on the day of receipt.

Test system

Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 μL
- Concentration: undiluted test substance
Duration of treatment / exposure:
Exposure period of 10 minutes (at 32 ± 1°C)
Duration of post- treatment incubation (in vitro):
2 hours (at 32 ± 1°C)
Number of animals or in vitro replicates:
Three corneas were selected for each group.
Details on study design:
PREPARATION OF CORNEAS
The corneal holder, MEM, and cMEM (MEM without phenol red) were warmed in a water bath or a low temperature incubator (set at 32°C) until use. The eyes were checked for unacceptable defects, such as opacity, scratches and neovascularization. There were no eyes exhibiting defects. Corneas were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling. Isolated corneas were mounted in corneal holders that consist of anterior and posterior compartments, which interface with the epithelial and endothelial sides of cornea, respectively. Both compartments of the corneal holder were filled with cMEM. The corneas were incubated for 1 hour in a low temperature incubator (set at 32°C, permissible range: 32°C ± 1°C, actual temperature: 31°C to 32°C). 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. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity was calculated according to the following equation.
Opacity = [(baseline LUX†) / (opacity of cornea LUX) – 0.9894] / 0.0251
†; Baseline LUX=1000 (The baseline was adjusted to1000 with cMEM.)
Corneas that had an initial opacity reading higher than 7 were not used. Nine corneas were chosen for the above experiment excluding those with high opacity.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
The following operations were performed in a draft chamber. Three corneas were selected for each group. The medium was removed from the anterior compartment and 750 μL of the negative control, the test substance and the positive control were introduced into the anterior compartment through dosing holes on the top surface of the chamber, and the holes were sealed (in the closed-chamber method). The holder was maintained in a horizontal position to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated for 10 minutes in a low temperature incubator (set at 32°C, permissible range: 32°C±1°C, actual temperature: 31°C to 32°C).

WASHING CORNEAS
After incubation, the control substances and the test substance were removed and the epithelium was washed three times with MEM. The corneas were washed once again because some test substance still remained. The corneas were given a final rinse with cMEM and were incubated for 2 hours in a low temperature incubator (set at 32°C, permissible range: 32°C±1°C, actual temperature: 31°C to 32°C).

OPACITY MEASUREMENT AND PERMEABILITY DETERMINATIONS
After the incubation period, each cornea was visually observed (e.g., tissue peeling, residual test substance, non-uniform opacity patterns). Medium was removed from both compartments and replaced with fresh cMEM and opacity determinations were performed. The medium of anterior compartments was removed and replaced with 1 mL of fluorescein sodium salt solution. Corneas were incubated in a horizontal position for 90 minutes in a low temperature incubator (set at 32°C, permissible range: 32°C±1°C, actual temperature: 31°C to 32°C). After the incubation period, the medium in the posterior compartments of each holder was removed and placed into a sampling tube. The optical density at 490 nm (OD490) of each sampling tube was measured using a spectrophotometer.

CALCULATION OF IN VITRO IRRITANCY SCORE
OPACITY
The final opacity for each individual cornea was calculated by subtracting the initial opacity reading from the post-treatment reading. The mean of the final opacity value of each treatment group was calculated. To calculate the in vitro irritancy score, values obtained by subtracting the mean final opacity of the negative control from that of the test substance or positive control were used for the calculation.

PERMEABILITY
The each values (OD490) was corrected for background value (OD490 value of cMEM only):
Permeability (OD490) = Each value (OD490) – Mean negative control value (OD490)

IN VITRO IRRITANCY SCORE
IVIS = Mean opacity value + (15 × Mean permeability OD490 value)
Individual IVIS = Individual opacity value + (15 × individual permeability OD 490 value)

DATA EVALUATION
DECISION CRITERIA
IVIS cut-off values for identifying the test substance as inducing serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below.
IVIS ≤ 3 was classified as UN GHS No Category,
IVIS > 3; ≤ 55 as No prediction can be made, and
IVIS > 55 as UN GHS Category 1.

ACCEPTANCE CRITERIA
The test is considered acceptable if:
(1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
(2) The negative control responses should result in opacity and permeability values below the upper limit of the laboratory historical range.

CRITERIA FOR RE-EXPERIMENT
Criteria for re-experiment are shown below; however, as none fell into the criteria, no re-experiment was performed in this study.
(1) Data are not obtained or reliability of the data is not confirmed due to an equipment failure.
(2) Apparent mistakes in the operation.
(3) Acceptability of the test does not meet the criteria.
(4) IVIS values vary among 3 corneas; predictions from 2 of the 3 corneas do not correspond to the mean of all 3 corneas or predictions from 1 of the 3 corneas do not correspond to the mean of all 3 corneas and the difference from the cut-off threshold (55) exceeds 10.
(a) When the results of a second testing run correspond to the prediction of the first testing run (based upon the mean IVIS value), the prediction of the second testing run should be accepted.
(b) When the results of a second do not correspond to the prediction from the first testing run (based upon the mean IVIS value), a third testing run should be conducted.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean In Vitro Irritancy Score
Value:
39.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table1 Summary of opacity, permeability, and in vitro irritancy score (IVIS)

Treatment

Mean opacity

Mean permeability

Mean in vitro irritancy score

Negative control

0.1

0.0282

0.5 

Kurimate

12.5

1.8063

39.1 

Positive control

81.1

2.6470 

120.4 

Appemdix 1 Individual opacity, permeability, and in vitro irritancy scores (IVIS)

Treatment Eyes Nos. Opacity Pereability in vitro irriteancy score (IVIS)
Negative control 1  -0.5 0.0199  -0.2 
  3  0.7 0.0427  1.3 
   0.0 0.0221  0.4 
Kurimate 6  12.4 1.9797  41.6 
  7  12.1 1.8822  39.8 
  8  12.9 1.5571  35.7 
Positive control 9  65.3 1.4107  85.9 
  14  90.4 3.4951  142.3 
  15  87.8 3.0351  132.8 

Appendix 2 Historical control data for BCOP studies

 Treatment Negative control      Positive control
   Permeability  Opacity  in vitro irritancy score    (IVIS)
 Range  <0.0590  <4.5  61.7 to 169.1
 Mean  0.0124  0.8  115.4
 n  42  42  39

The above data range of the control substances were obtained by collecting all data over the period of December 2015 to July 2017.

Applicant's summary and conclusion

Interpretation of results:
other: No prediction of eye irritation can be made
Conclusions:
Since Kurimate induced an IVIS > 3 and ≤ 55, no prediction could be made for eye irritation or serious eye damage.
Executive summary:

The opacity, permeability andin vitroirritancy scores (IVIS) of Kurimate and the control substances are shown in Table 1 and Appendix 1.

The eye irritancy potential of Kurimate was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test). The test method used isolated corneas from the eyes of freshly slaughtered cattle.  The corneas damaged by the test substance were assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a spectrophotometer, respectively.  Both measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the eye irritancy of the test substance. 

Negative control values for opacity and permeability were below the upper limit of the test facility historical range.  Therefore, the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (N,N-dimethylformamide) was 120.4 and 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.

Kurimate induced opacity and increased permeability.  This resulted in a mean IVIS of 39.1 after 10 minutes of treatment.  Since Kurimate induced an IVIS > 3 and ≤ 55, no prediction could be made for eye irritation or serious eye damage.