Reaction products of neodecanoic acid and titanium (IV) isopropoxide

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed on 13 November 2009.

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:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

no guideline followed

Guideline:

other: The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential.

Deviations:

no

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)

Test material

Test material information

Constituent 1

Reference substance name:

E0286P/040A

IUPAC Name:

E0286P/040A

Details on test material:

Sponsor's identification: E0286P/040A
Description : amber coloured viscous liquid
Batch number : E0286P/040A/01
Date received : 20 July 2009
Storage conditions: room temperature in the dark

Test animals / tissue source

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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-1000 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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

For the purpose of the study the test material was used as supplied.

Controls:

other: Two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).

Amount / concentration applied:

The test material was used undiluted as supplied. A volume of 0.1 ml of the test material was applied as evenly as possible to 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.

The thickness of the cornea was measured using an ultrasonic pachymeter. 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.

The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 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.
The test material was used undiluted as supplied. A volume of 0.1 ml of the test material was applied as evenly as possible to 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).

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:

cornea opacity score

Remarks:

Corneal Opacity

Basis:

other: Maximum score

Time point:

other: Maximum score

Max. score:

1

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Maximum score for Corneal Opacity 1

Results 2

Irritation parameter:

cornea opacity score

Remarks:

Area of corneal opacity

Basis:

other: Maximum score

Time point:

other: Maximum score

Max. score:

3

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Maximum score for Area of corneal opacity 3

Results 3

Irritation parameter:

cornea opacity score

Remarks:

Corneal Swelling (%)

Basis:

other: Mean score

Time point:

other: 60 Minutes

Score:

13.8

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Mean score @ 60 Minutes 13.8 %

Results 4

Irritation parameter:

cornea opacity score

Remarks:

Corneal Swelling (%)

Basis:

other: Mean score

Time point:

other: 120 Minutes

Score:

35.3

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Mean score @ 120 Minutes 35.3 % which meets or exceeds cut-off value indicating a severe ocular irritant

Results 5

Irritation parameter:

cornea opacity score

Remarks:

Corneal Swelling (%)

Basis:

other: Mean score

Time point:

other: 240 Minutes

Score:

58

Reversibility:

not specified

Remarks:

N/A

Remarks on result:

other: Mean score @ 240 Minutes 58.0 % which meets or exceeds cut-off value indicating a severe ocular irritant

Results 6

Irritation parameter:

cornea opacity score

Remarks:

Condition of Corneal Epithelium

Basis:

other: Maximum score

Time point:

other: Maximum score

Reversibility:

not specified

Remarks on result:

other: Sloughing was seen which 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 eyes. 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. The condition of the corneal epithelium following treatment is given in Table 4.
Corneal swelling of the test eyes during the study period was considerably greater than to that observed in the control eyes over the same period.
Sloughing of the corneal epithelium was noted in two test eyes. The condition of the corneal epithelium of one test eye and the control eyes appeared normal during the study period.

Fluorescein Uptake
Individual scores for fluorescein uptake are given in Table 5.
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.

Any other information on results incl. tables

Interpretation of Results

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*	REET Cut-Off Value
Maximum Corneal Opacity (Corneal Cloudiness x Area)	> or = 4
Maximum Fluorescein Uptake (Intensity x Area)	> or = 4
Mean Corneal Swelling (mins): 60, 120, 240	> or = 25%
Corneal Epithelium Observations	Any with pitting, mottling or sloughing

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

(mean corneal thickness post – treatment) – (mean corneal thickness post equilibration) x 100

Mean corneal thickness post equilibration

A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 minutes post treatment observation periods.

A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.

*= Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant.

Table1              Individual Scores for Corneal Opacity

	Test Eyes												Control Eyes
Chamber Number	1A				3A				5A				2A				4A
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	0	0	0	1	0	1	1	1	0	1	1	1	0	0	0	0	0	0	0	0
Area of Corneal Opacity	0	0	0	1	0	1	1	1	0	2	3	3	0	0	0	0	0	0	0	0

Table2              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	1A	408	450	397	386	398	407.8	434	468	443	425	460	446.0	435	521	443	457	497	470.6	460	630	453	457	523	504.6
	3A	426	423	401	420	422	418.4	441	587	425	551	529	506.6	455	657	431	592	612	549.4	463	677	444	682	693	581.0
	5A	439	437	441	437	443	439.4	502	455	575	621	631	556.8	736	459	640	705	703	648.6	721	471	699	756	753	680.0
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	2A	389	353	362	383	362	369.8	386	337	365	373	359	364.0	369	343	345	360	352	353.8	363	329	343	355	354	348.8
	4A	421	374	373	404	398	394.0	411	376	372	403	405	393.4	401	367	372	390	384	382.8	400	369	355	383	371	375.6

oc=    Optical centre

Table3              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
1A	Post equilibration	347.8	N/A	3A	Post equilibration	368.0	N/A	5A	Post equilibration	397.4	N/A
	60 Post treatment	407.8	17.3		60 Post treatment	418.4	13.7		60 Post treatment	439.4	10.6
	120 Post treatment	446.0	28.2		120 Post treatment	506.6	37.7		120 Post treatment	556.8	40.1
	180 Post treatment	470.6	35.3		180 Post treatment	549.4	49.3		180 Post treatment	648.6	63.2
	240 Post treatment	504.6	45.1		240 Post treatment	581.0	57.9		240 Post treatment	680.0	71.1

 

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 13.8% Mean corneal swelling 2 hours following treatment 35.3%+ Mean corneal swelling 4 hours following treatment 58.0%+
2A	Post equilibration	353.2	N/A	4A	Post equilibration	384.4	N/A
	60 Post treatment	369.8	4.7		60 Post treatment	394.0	2.5	Control Eyes
	120 Post treatment	364.0	3.1		120 Post treatment	393.4	2.3	Mean corneal swelling 1 hour following treatment 3.6% Mean corneal swelling 2 hours following treatment 3.8% Mean corneal swelling 4 hours following treatment 0.0%
	180 Post treatment	353.8	0.2		180 Post treatment	382.8	0.0
	240 Post treatment	348.8	0.0		240 Post treatment	375.6	0.0

a=     % Corneal swelling =

N/A=  Not applicable

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

Table4              Corneal Epithelium Condition

Test Eyes
Chamber Number	Time After Treatment (minutes)
	60	120	180	240
1A	Normal	Normal	Normal	Normal
3A+	Normal	Sloughing	Sloughing	Sloughing
5A+	Normal	Sloughing	Sloughing	Sloughing

 

Control Eyes
Chamber Number	Time After Treatment (minutes)
	60	120	180	240
2A	Normal	Normal	Normal	Normal
4A	Normal	Normal	Normal	Normal

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

Table5              Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

	Test Eyes			Control Eyes
Chamber Number	1A	3A	5A	2A	4A
Intensity of Fluorescein Uptake	1	1	2	0	0
Area of Fluorescein Uptake	2	2	3	0	0

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 was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.5°C within the superfusion chamber. 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
Cldy	Area	Int	Area	60 mins	120 mins	240 mins	60 mins	120 mins	240 mins
1	3	2	3	13.8	35.3+	58.0+	3.6	2.7	0.0	Sloughing+

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.

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