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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed on the 05 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was designed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye.
The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential. The preferred species of choice is the rabbit. The assay has undergone inter-laboratory validation and has been shown to reliably detect test materials that are negligible, or moderate to severe ocular irritants.
The strain of rabbit used in these laboratories has been shown to produce satisfactory responses using known ocular-irritants and non-ocular irritants during in-house validation (see attached Appendix 3). The results of the study are believed to be of value in predicting the ocular irritancy potential of the test material in man.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 15-09-2009 Date of Signature: 26-11-2009

Test material

Constituent 1
Reference substance name:
1,3-Cyclohexanedione
IUPAC Name:
1,3-Cyclohexanedione
Details on test material:
Sponsor's identification: 1,3-Cyclohexanedione
Description : pale beige solid
Batch number : 1091224
Date received : 24 November 2009
Expiry date : 18 May 2010
Storage conditions: approximately 4°C in the dark
A Certificate of Analysis is given in attached Appendix 4.

Test animals / tissue source

Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Selection of Eyes
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1% w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope (Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using the DGH-55 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes (Appendix 1).

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
For the purpose of the study the test material was used as supplied.
Controls:
other: two additional eyes remained untreated for control purposes.
Amount / concentration applied:
A volume of 0.1 ml of the test material, which was found to weigh approximately 63 mg (as measured by gently compacting the required volume into an adapted syringe) was sprinkled as evenly as possible over the surface of the cornea.
Duration of treatment / exposure:
After ten seconds the test material was washed off the cornea using a minimum of 20 ml of saline solution (approximately 32°C).
Observation period (in vivo):
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
Number of animals or in vitro replicates:
Three eyes were treated with test material, two additional eyes remained untreated for control purposes.
Details on study design:
Test Material Administration
Three eyes were treated with test material, 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.
A volume of 0.1 ml of the test material, which was found to weigh approximately 63 mg (as measured by gently compacting the required volume into an adapted syringe) was sprinkled as evenly as possible over the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 ml of saline solution (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.

Observations
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment, according to the numerical evaluation given in Appendix 2 adopted from Advances in Modern Toxicology: Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749-815.
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation given in attached Appendix 2. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Maximum Corneal Opacity (corneal cloudiness x area)
Basis:
other: Maximum Score
Time point:
other: Maximum Score
Score:
4 - 8
Max. score:
8
Reversibility:
not specified
Remarks on result:
other: Please see Table 1 for full results. The scores were 4+ to 8+ where + = Meets or exceeds cut-off value indicating a severe ocular irritant.
Irritation parameter:
cornea opacity score
Remarks:
Mean corneal swelling 1 hour following treatment.
Basis:
mean
Time point:
other: 60 Minutes
Score:
43.7
Max. score:
56
Reversibility:
other: Not applicable
Remarks on result:
other: Mean corneal swelling 1 hour following treatment 43.7%+ (+ = meets or exceeds cut-off value and therefore indicates a severe eye irritant)
Irritation parameter:
cornea opacity score
Remarks:
Mean corneal swelling 2 hour following treatment.
Basis:
mean
Time point:
other: 120 Minutes
Score:
65.9
Max. score:
77.2
Reversibility:
other: Not applicable
Remarks on result:
other: Mean corneal swelling 2 hour following treatment 65.9 %+ (+ = meets or exceeds cut-off value and therefore indicates a severe eye irritant)
Irritation parameter:
cornea opacity score
Remarks:
Mean corneal swelling 4 hour following treatment 97.8 % + (+ = meets or exceeds cut-off value and therefore indicates a severe eye irritant)
Basis:
mean
Time point:
other: 240 Minutes
Score:
97.8
Max. score:
110.8
Reversibility:
other: Not applicable
Remarks on result:
other: Mean corneal swelling 4 hour following treatment 97.8 %+ (+ = meets or exceeds cut-off value and therefore indicates a severe eye irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 6A
Time point:
other: 60 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 8A
Time point:
other: 60 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 10A
Time point:
other: 60 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 6A
Time point:
other: 120 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 8A
Time point:
other: 120 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 10A
Time point:
other: 120 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 6A
Time point:
other: 240 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 8A
Time point:
other: 240 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Sloughing+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Corneal Epithelium Condition
Basis:
other: Chamber Number 10A
Time point:
other: 240 Minutes
Reversibility:
other: Not applicable
Remarks on result:
other: Corneal Epithelium Condition Pitting+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritation parameter:
other: Maximum Fluorescein Uptake (intensity x area)
Basis:
other: Maximum Fluorescein Uptake
Time point:
other: 240 Minutes
Score:
8
Max. score:
8
Reversibility:
other: Not applicable
Remarks on result:
other: Maximum Fluorescein Uptake (intensity x area) 8+ (+ = Meets or exceeds cut-off value indicating a severe ocular irritant)
Irritant / corrosive response data:
Corneal Opacity
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test animals 60 minutes after dosing with moderate loss of transparency noted at subsequent observations.
No corneal effects were noted in the control eyes during the study period.

Corneal Thickness and Condition
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3.
Corneal swelling of the test eyes during the study period was considerably greater than that observed in the control eyes over the same period.
The condition of the corneal epithelium following treatment is given in Table 4.
Pitting of the corneal epithelium was noted in test eyes 60, 120 and 180 minutes after dosing with pitting or sloughing 240 minutes after dosing.
The condition of the corneal epithelium of the control eyes appeared normal during the study period.

Fluorescein Uptake
Individual scores for fluorescein uptake are given in Table 5.
Moderate fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.
Other effects:
Not applicable

Any other information on results incl. tables

Table 1              Individual Scores for Corneal Opacity

 

Test Eyes

Control Eyes

Chamber Number

6A

8A

10A

7A

9A

Time After Treatment (minutes)

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

Degree of Corneal Opacity

1

2

2

2

1

2

2

2

1

2

2

2

0

0

0

0

0

0

0

0

Area of Corneal Opacity

4

4

4

4

4

4

4

4

4

4

4

4

0

0

0

0

0

0

0

0

Maximum Corneal Opacity (corneal cloudiness x area)

4+

8+

8+

8+

4+

8+

8+

8+

4+

8+

8+

8+

0

0

0

0

0

0

0

0

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

Table 2              Individual Measurements for Corneal Thickness (µm)

Test Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

Chamber Number

6A

590

557

559

589

558

570.6

660

616

645

676

604

640.2

714

665

710

712

672

694.6

747

706

758

759

709

735.8

8A

511

476

489

493

489

491.6

618

576

580

601

605

596.0

691

662

657

665

683

671.6

758

720

752

748

724

740.4

10A

616

564

574

597

583

586.8

698

647

636

697

656

666.8

759

696

728

747

729

731.8

809

762

789

808

797

793.0

 

Control Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

Chamber Number

7A

406

395

383

390

396

394.0

401

388

380

381

388

387.6

401

382

383

376

391

386.6

386

373

366

367

376

373.6

9A

406

403

377

397

396

395.8

396

391

369

378

388

384.4

393

390

367

374

383

381.4

399

394

361

377

394

385.0

oc=   Optical centre

Table 3              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

6A

Post equilibration

386.0

N/A

8A

Post equilibration

385.8

N/A

10A

Post equilibration

376.2

N/A

60 Post treatment

570.6

47.8

60 Post treatment

491.6

27.4

60 Post treatment

586.8

56.0

120 Post treatment

640.2

65.9

120 Post treatment

596.0

54.5

120 Post treatment

666.8

77.2

180 Post treatment

694.6

79.9

180 Post treatment

671.6

74.1

180 Post treatment

731.8

94.5

240 Post treatment

735.8

90.6

240 Post treatment

740.4

91.9

240 Post treatment

793.0

110.8

 

Control 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

Test Eyes

Mean corneal swelling 1 hour following treatment 43.7%+

Mean corneal swelling 2 hours following treatment 65.9%+

Mean corneal swelling 4 hours following treatment 97.8%+

7A

Post equilibration

381.6

N/A

9A

Post equilibration

365.8

N/A

60 Post treatment

394.0

3.2

60 Post treatment

395.8

8.2

Control Eyes

120 Post treatment

387.6

1.6

120 Post treatment

384.4

5.1

Mean corneal swelling 1 hour following treatment 5.7%

Mean corneal swelling 2 hours following treatment 3.3%

Mean corneal swelling 4 hours following treatment 2.6%

180 Post treatment

386.6

1.3

180 Post treatment

381.4

4.3

240 Post treatment

373.6

0.0

240 Post treatment

385.0

5.2

a=     % Corneal swelling =

N/A=  Not applicable

+ =    Meets or exceeds cut-off value and therefore indicates a severe eye irritant

Table 4              Corneal Epithelium Condition

Test Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

6A

Pitting+

Pitting+

Pitting+

Pitting+

8A

Pitting+

Pitting+

Pitting+

Sloughing+

10A

Pitting+

Pitting+

Pitting+

Pitting+

 

Control Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

7A

Normal

Normal

Normal

Normal

9A

Normal

Normal

Normal

Normal

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

Table 5              Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

 

Test Eyes

Control Eyes

Chamber Number

6A

8A

10A

7A

9A

Intensity of Fluorescein Uptake

2

2

2

0

0

Area of Fluorescein Uptake

4

4

4

0

0

Maximum Fluorescein Uptake
(intensity x area)

8+

8+

8+

0

0

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

Please also see: attached Appendix 2 Method for Evaluation of Ocular Irritation by Slit-Lamp Biomicroscopic Examination - McDonald - Shadduck Score System, Appendix 3 In-house Validation Data & Appendix 4 Certificate of Analysis

Applicant's summary and conclusion

Interpretation of results:
highly irritating
Remarks:
Migrated information Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo. Criteria used for interpretation of results: expert judgment
Conclusions:
Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

Introduction. 

A study was performed to assess the ocular irritancy potential of the test material 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 material in man. 

Methods. 

0.1 ml of the test material, which was found to weigh approximately 63 mg, was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.7°C within the superfusion chamber. One chamber was slightly outside the specified range (32°C ± 1.5°C) at 30.3°C but this deviation was considered not to affect the purpose or integrity of the study. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).

Results.

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

Corneal Opacity

Fluorescein Uptake

Corneal Swelling (%)

Condition of Corneal Epithelium

Test Eyesa

Control Eyesb

Cldyx Area

Intx Area

60mins

120 mins

240 mins

60 mins

120 mins

240 mins

8+

8+

43.7+

65.9+

97.8+

5.7

3.3

2.6

pitting/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 fluorescein uptake

mins= 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 material was considered to have the potential to cause severe ocular irritancy in vivo.