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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 January - 19 January 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:
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
Type:
impurity
Type:
impurity
Test material form:
liquid: volatile
Details on test material:
Batch No: 57161235
Storage: Room temperature (approx. 20°C)

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: ABP, Perth, PH1 3XB
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to test facility was on the same day as slaughter. Cold packs were used to keep the contents cool.
- Time interval prior to initiating testing: Corneas were prepared and used for testing on the day of collection
- indication of any existing defects or lesions in ocular tissue samples: Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use.Any eyes showing defects were rejected from further use.
- Indication of any antibiotics used: Not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration Test Item: 100 %
NEGATIVE CONTROL: Physiological saline
- Amount(s) applied: 750 µ
- Concentration: Sodium Chloride 0.9%, w/v
POSITIVE CONTROL : Ethanol
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration: 100 %, w/v in physiological saline
Duration of treatment / exposure:
10 min ± 1 min
Duration of post- treatment incubation (in vitro):
2h ± 5 min
Number of animals or in vitro replicates:
Three Corneas per test item and control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of ca 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use. Prior to mounting corneas the width of the cornea was measured using a ruler.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were then mounted, epithelial side forward, into pre-warmed corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed EMEM (Eagles Minimum Essential Medium) without phenol red. The posterior chamber was filled first to encourage the cornea to return to its original curvature, and care was taken to avoid the introduction of bubbles into the medium. Holders were then allowed to equilibrate for >1 h in an incubator.
Unless otherwise stated, all incubations of corneas were conducted in an incubator set to maintain 32°C (actual temperatures have been recorded in the raw data). At the end of the equilibration period, the medium in both chambers was replaced with fresh, EMEM without phenol red. Baseline opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Damaged corneas (opacity >7 opacity units) were rejected from further use.

TREATMENT METHOD: closed chamber. Prior to dosing, cornea holders were tipped forward to avoid contact between the dosing solution and the corneal epithelium. Vinyl propionate (750 µL) was applied to the surface of three corneas using a syringe. The negative control, physiological saline (750 µL), and the positive control, ethanol (750 µL), were also applied to the surface of three corneas each, undiluted, using a syringe. The anterior chambers were then sealed with tape. To begin exposure, the corneal holders were tilted to a horizontal position, taking care to ensure that the entire epithelial surface was covered with the dosing solution. The dosed units were then returned to the incubator for 10 min ± 1 min.

REMOVAL OF TEST SUBSTANCE
EMEM with and without phenol red were pre-warmed prior to use. Following the exposure incubation, the test items and control solutions were removed from the anterior chambers through the holes using a vacuum pump. Corneas were then rinsed three times with EMEM with phenol red (ca 5 mL per rinse), removing the EMEM each time, then rinsed once with EMEM without phenol red (ca 5 mL), which was also removed. Finally, both chambers were refilled with EMEM without phenol red, and any bubbles introduced into the medium were removed.

POST-INCUBATION PERIOD: 2 h ± 5 min.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Gross damage or other changes were observed by visual assessment.

- Corneal pemeability:
Following the measurement of opacity, the EMEM in both chambers of the cornea holder was removed. Posterior chambers were refilled with EMEM without phenol red and sealed. Sodium fluorescein solution (ca 1 mL, 4 mg/mL (liquids method) or 5 mg/mL (solids method) in EMEM without phenol red) was added to the anterior chambers. Cornea holders were rotated to the horizontal position, and incubated for 90 min ± 5 mins in an incubator set to maintain a temperature of 32°C (actual temperatures have been recorded in the raw data).

Due to an equipment malfunction post dose opacity measurements for corneas 22 and 23 (dosed with Vinyl propionate) were rejected after the addition of sodium fluorescein solution. After ca 5 mins of incubation with sodium fluorescein these corneas were removed from the incubator and rinsed 5 times (front and back) with EMEM without phenol red. The chambers were filled with EMEM without phenol red and post dose opacity was measured again. Following the repeat opacity measurements the corneas were refilled with 1 mL of 4 mg/mL sodium fluorescein and exposure was began as described above. For a discussion of the impact of this on the study results, see results section

At the end of the permeability test, EMEM from the posterior chambers was collected. These samples were stored overnight in a refrigerator set to maintain a temperature of 4°C.
Following overnight storage, triplicate aliquots from each sample (380 µL) were transferred into a 96 well plate, and analysed with a Multiskan Spectrum plate reader at 490 nm. Three blank wells containing EMEM without phenol red (380 µL) were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluorescein, 10 µg/mL). Using the standard curve prepared previously these two quality control samples gave acceptable results, of ca 10 µg/mL, when the plate was read.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = Opacity change + (15 x A490 Value)

DECISION CRITERIA:
In accordance with OECD Test Guideline No. 437, irritancy was assigned on the basis of in vitro irritancy scores (IVIS), calculated from the opacity and permeability value for each cornea, as follows:

IVIS<=3: not classified according to GHS/CLP
3IVIS>55: Classified as Eye Damaging Category 1 according to GHS/CLP

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
The following acceptance criteria have been met, and therefore the test is considered to be acceptable
Run / experiment:
Single experiment
Value:
25
Vehicle controls valid:
yes
Remarks:
IVIS: 0.0
Positive controls valid:
yes
Remarks:
IVIS: 63.27
Remarks on result:
not determinable
Remarks:
No Prediction can be made
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean post treatment opacity of 2.47 (historical values range 3.56 +/- 1.82) / mean permeability corrected for blank of 0.003 (historical values range 0.016 +/- 0.016). The classification achieved was “No Category”.
- Acceptance criteria met for positive control: yes, mean IVIS 63.27 (historical range: Mean +/- 2SD =42.28 to 98.49). The classification achieved was “Category 1".
- Test item treatement: The BCOP results were similar for all corneas and are presented in full in Table 1 (see Any other information on results incl. tables section).

For Vinyl propionate, two of the three replicate corneas (corneas 22 and 23) were treated with sodium fluorescein prior to the measurement of post-exposure opacity, in error. This error was noticed after ca 5 min, and rectified. Opacity was then measured, and then the procedure followed as for the other replicate (cornea 21). However, it was noted in the raw data that this brief pre-treatment with sodium fluorescein prior to opacity measurement slightly stained the corneas. This is apparent from the opacity change values for these two corneas (13.06 and 11.51), which are higher than their correctly treated counterpart (8.03). The result of this is that a higher IVIS score than would have otherwise been calculated. There was no clear difference in permeability for these three samples. However, the increased opacity change observed for corneas 22 and 23 compared to what would have been expected, has not affected the prediction outcome. The result of “no prediction can be made” would still have been achieved even with lower opacity results were achieved for these corneas.

Any other information on results incl. tables

Table 1                         BCOP Results for Controls and Vinyl Propionate (n=3)

 Treatment   

Opacity Post Dose

(Opacity Units, Uncorrected)

  

Corrected* Opacity Change

(Opacity Units)

 
Permeability (A490), Corrected for Blank and Dilution Only  

   

Permeability (A490), Corrected*

  

IVIS

 

IVIS (Mean)

 

UN GHS Category**

Physiological Saline (Negative Control)

5.69

6.39

7.65

  -171

0.26

1.45

0.003

0.005

0.002

0.000

0.002

-0.001 

  -1.72

0.29

1.43

 0.00

No Category

Ethanol (PositiveControl)

37.56

38.57

39.60

30.81

30.75

33.17

1.716

1.915

2.718

1.713

1.912

2.715

56.50

59.42

73.88

63.27 

 Category 1

 Vinyl Propionate

13.85

18.65

17.95

 8.03

13.06

11.51

0.492

1.515

0.836 

0.489

1.511

0.832

15.37

35.73***

24.00***

25.03***

 No prediction can be made

* Corrected for Negative Control

**Classifications from OECD Guidelines for the Testing of Chemicals No. 437 (2013)

*** IVIS calculated from corneas that had post dose opacity measurements after the addition and subsequent removal of sodium fluorescein. See Section 6.8.3 for details.

Applicant's summary and conclusion

Interpretation of results:
other: no prediction could be made
Remarks:
This endpoint study record is part of a Weight of Evidence approach comprising two in vitro studies. Both are evaluated in a combined approach to determine the classification
Conclusions:
In conclusion, no prediction could be made for Vinyl propionate (CAS 105-38-4) using the BCOP assay according to the UN GHS classifications and the CLP classification system.
Executive summary:

The objective of this study was to evaluate the ocular irritation potential of Vinyl propionate (CAS 105-38-4) using the Bovine Corneal Opacity and Permeability (BCOP) assay (OECD guideline 437).

Corneas were dissected from freshly obtained cow eyes, the cornea diameter was measured and they were mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated as follows:

Vinyl propionate (750 µL), physiological saline or ethanol as negative and positive controls, respectively, were each applied to three corneas. Following exposure for 10 min ± 1 min, the test or control items were rinsed off and corneas underwent a recovery period of 2 h ± 5 min

The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The results are summarised as follows:

The mean IVIS Score for Vinyl propionate was 25.0

In conclusion, no prediction could be made for Vinyl propionate (CAS 105-38-4) using the BCOP assay according to the UN GHS classifications and the CLP classification system.