Cyclohexane-1,3-dione

Eye irritation

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

Test material information

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

Results

Results 1

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.

Results 2

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)

Results 3

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)

Results 4

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)

Results 5

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)

Results 6

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)

Results 7

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)

Results 8

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)

Results 9

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)

Results 10

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)

Results 11

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)

Results 12

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)

Results 13

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)

Results 14

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 Eyes^a			Control Eyes^b
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.