Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 11 February 2016 Experimental completion date: 11 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: The Rabbit Enucleated Eye Test
Version / remarks:
The Rabbit Enucleated Eye Test has been included in evaluations of the validity of eye irritancy test methods, but is not currently considered to be a validated 'stand-alone' test method. No formally adopted test guideline is available, but a protocol has been proposed which was based upon the method detailed in this document (ICCVAM, 2006 and 2009).

A positive result in the rabbit enucleated eye test is accepted within the EU as an indication of severe eye irritancy potential without the need for confirmation in a rabbit eye irritation test (European Chemicals Bureau, 2006).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
other: yellow semi-solid

Test animals / tissue source

Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals were 12 to 20 weeks old. Rabbits that have previously been used in skin or eye irritation studies at the test facility may. be used as eye donors. If the donor animals have been used in an eye irritation study, only the untreated eyes were used.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
For the purpose of the study the test item was used as supplied.
Controls:
yes
yes, concurrent no treatment
Amount / concentration applied:
Three eyes were treated with the test item. Two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.

The test item was used undiluted as supplied.
A volume of 0.1 mL of the test item was applied as evenly as possible to the surface of the cornea
Duration of treatment / exposure:
After ten seconds the test item was washed off the cornea using a minimum of 20 mL of 0.9% saline solution (pre-warmed to approximately 32 °C).

Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye.

The untreated eyes were similarly washed and used for control purposes.
Duration of post- treatment incubation (in vitro):
Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye.
Number of animals or in vitro replicates:
5 eyes
Details on study design:
Please see below

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
Mean Corneal Swelling
Run / experiment:
60 runs
Value:
31.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean Corneal Swelling
Run / experiment:
120 runs
Value:
54.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean Corneal Swelling
Run / experiment:
240 runs
Value:
90
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Corneal Opacity

Individual scores for corneal opacity are given in the attached Appendix 1.

Some loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study period.

Corneal Thickness

Individual and mean measurements for corneal thickness are given in the attached Appendix 2. The determination of corneal swelling is given in Table 1 and Table 2. The calculated mean corneal swellings are given in Table 3.

Corneal swelling of the test eyes was considerably greater than that observed in the control eyes over the same period and exceeded the 2.25% cut-off value.

Corneal Condition

The condition of the corneal epithelium following treatment is given in the attached Appendix 3.

Sloughing of the corneal epithelium was noted in test eyes. The condition of the corneal epithelium of the control eyes appeared normal during the study.

Fluorescein Uptake

Individual scores for fluorescein uptake are given in the attached Appendix 4.

Fluorescein uptake was noted in the test eyes 240 minutes following test item application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Table 1            Determination of Corneal Swelling-Test Eyes

 

Chamber Number

 

Observation Period (minutes)

Mean Corneal Thickness(µm)

 

Corneal Swelling(%)a

 

Chamber Number

 

Observation Period (minutes)

Mean Corneal Thickness(µm)

 

Corneal Swelling(%)a

 

Chamber Number

 

Observation Period (minutes)

Mean Corneal Thickness(µm)

Corneal Swelling(%)a

 

 

 

 

 

 

IA

Post equilibration

 

355.8

 

na

 

 

 

 

 

 

3A

Post equilibration

 

400.0

 

na

 

 

 

 

 

 

5A

Post equilibration

 

414.2

 

na

60 Post treatment

 

520.2

 

46.2

60 Post treatment

 

514.0

 

28.5

60 Post treatment

 

493.8

 

19.2

120 Post treatment

 

643.8

 

80.9

120 Post treatment

 

578.4

 

44.6

120 Post treatment

 

573.6

 

38.5

180 Post treatment

 

771.6

 

116.9

180 Post treatment

 

612.8

 

53.2

180 Post treatment

 

633.8

 

53.0

240 Post treatment

 

854.0

 

140.0

240 Post treatment

 

655.4

 

63.9

240 Post treatment

 

688.6

 

66.2

a = % corneal swelling = (mean corneal thickness post-treatment) - (mean corneal thickness post equilibration) / mean corneal thickness post equilibrium x 100

Na = Not applicable

Table2            Determination of Corneal Swelling - Control Eyes

 

ChamberNumber

 

Observation Period (minutes)

 

Mean Corneal Thickness (µm)

 

Corneal Swelling(%)a

 

Chamber Number

 

Observation Period(minutes)

 

Mean Corneal Thickness (µm)

 

Corneal Swelling(%)a

 

 

 

 

 

 

2A

 

Post equilibration

 

353.0

 

na

 

 

 

 

 

 

4A

 

Post equilibration

 

403.8

 

na

 

60 Post treatment

 

368.4

 

4.4

 

60 Post treatment

 

449.8

 

11.4

 

120 Post treatment

 

356.4

 

1.0

 

120 Post treatment

 

472.6

 

17.0

 

180 Post treatment

 

350.4

 

0.0

 

180 Post treatment

 

468.4

 

16.0

 

240 Post treatment

 

348.6

 

0.0

 

240 Post treatment

 

466.6

 

15.6

a = % corneal swelling = (mean corneal thickness post-treatment) - (mean corneal thickness post equilibration) / mean corneal thickness post equilibrium x 100

Na = Not applicable

Table3            Mean Corneal Swelling

 

 

Time After Treatment

 

60 minutes

 

120 minutes

 

240 minutes

 

Test Eyes

 

31.3+

 

54.7+

 

90.0+

 

Control Eyes

 

7.9

 

9.0

 

7.8

+ = Meets or exceeds cut-off value indicating a severe ocular irritant

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Following assessment of the data for all endpoints, the test item was considered to have the potential to cause severe ocular irritancy in viva.
Executive summary:

Introduction

 Itis a legal and ethical duty under the Animals (Scientific Procedure) Act 1986 that, in the interest of animal welfare, the unnecessary use of animals is avoided, and that any testing which is likely to produc esevere responses in animals is minimized.

 Therefore, before in vivo irritation testing is performed, all existing information on the test item, or its analogues should be reviewed. Available information indicated that the test item had the potential to produce severe effects in a rabbit eye and to confirm this initial assessment, a Rabbit Enucleated Eye Test (REET) was performed.

 

This step-wise procedure is in accordance with OECD Test Guideline 405,UK Home Office regulations and Envigo Research Limited Ethical Testing Strategy.

A study was performed to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test item in man.

 

Method

 0.1 mL of the test item was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ±1.5 °C within the superfusion chamber. A further two enucleated eyes remained untreated for control purposes.

Results

 Maximal ocular irritation observations recorded for the test eyes were as follows:

 

Corneal Opacity

FluoresceinUptake

Mean Corneal Swelling(%)

 

Condition of Corneal Epithelium

Test Eyes a

Control Eyes b

Cldy x Area

Int x Area

60

ruins

120

ruins

240

ruins

60

ruins

120

ruins

240

ruins

3

4+

31.3+

54.7+

90.0+

7.9

9.0

7.8

sloughing+

 

  

a=          For each time point the swelling recorded is the mean of three eyes

b=         For each time point the swelling recorded is the mean of two eyes

Cldy =   Corneal cloudiness

Int=       Intensity of fluoresceinuptake

mms = Minutes following treatment

+=         Meets or exceeds cut-off value indicating a severe ocular irritant


 

Conclusion

 Following assessment of the data for all endpoints the test item was considered to have the potential to cause severe ocular irritancy in vivo.