Petroleum, synthetic, hydrodesulfurized full-range coker

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed on 10 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

no guideline available

Deviations:

not applicable

Principles of method if other than guideline:

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 items that are negligible, or moderate to severe ocular irritants.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Crude oil (petroleum) with recycled naphtha, distilled, cracked, hydrotreated and hydrodesulfurized

EC Number:

700-919-3

Cas Number:

1379524-03-4

Molecular formula:

C3H8 to C[>100]H[>202]. Also some organic sulfur & nitrogen compounds - refer to analytical reports.

IUPAC Name:

Crude oil (petroleum) with recycled naphtha, distilled, cracked, hydrotreated and hydrodesulfurized

Test material form:

liquid: viscous

Details on test material:

Sponsor's identification: Hamaca Synthetic Crude Oil composite sample (671/672/675)
Description : black viscous liquid
Batch number : not supplied
Purity : not supplied
Date received : 31 May 2012
Expiry date : 31 May 2014
Storage conditions: room temperature in the dark
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

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:

10 seconds

Observation period (in vivo):

240 minutes

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Pre-Test Procedures
Superfusion Chamber
The water heating circulator (Julabo MP5, Jencons (Scientific) Ltd., Leighton Buzzard, Beds, UK), was adjusted so that the temperature of the water flowing through the water jacket of the superfusion apparatus, gave a stable temperature, of 32 ±1.5°C, within the chambers of the apparatus. A peristaltic pump (205S/BA, Watson Marlow Ltd, Falmouth, Cornwall; UK) was used to supply saline solution at a flow rate of 0.15 to 0.4 ml/minute (at approximately 32°C) into the rear of each chamber of the apparatus in order to irrigate the surface of the cornea.
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-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 (Appendix 1).
Enucleation of Eyes
The donor rabbits were sacrificed by intravenous administration of an overdose of sodium pentobarbitone. Immediately afterwards, two to three drops of saline solution (approximately 32°C) were applied to the cornea to prevent desiccation during excision. The eye was then carefully removed, positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber adjusted so that saline solution was allowed to irrigate the surface of the cornea. The eyes were then allowed to equilibrate for approximately thirty minutes. Following the equilibration period, the eyes were re examined to ensure they had not been damaged during excision. Corneal thickness was also measured using the ultrasonic pachymeter. Any eyes in which the corneal swelling was greater than 10% relative to the pre-enucleation measurement, or in which the cornea was stained with fluorescein, were rejected. The post equilibration corneal thickness values for each eye were recorded (Appendix 1).
PROCEDURE
Test Item Administration
Three eyes were treated with 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. 240 minut. After ten seconds the test item 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 (attachment1) 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 Appendix 2 (attachment 1). 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:

swelling

Basis:

other: Test

Time point:

other: Maximal score at 60, 120 and 210 minutes post dosing

Score:

10.1

Max. score:

25

Reversibility:

other: not applicable

Remarks on result:

other: Condition of Corneal Epithelium: Normal

Results 2

Irritation parameter:

cornea opacity score

Remarks:

swelling

Basis:

other: Control

Time point:

other: Maximal score at 60, 120 and 240 minutes post dosing

Score:

9.2

Max. score:

25

Reversibility:

other: not applicable

Remarks on result:

other: Condition of Corneal Epithelium: Normal

Results 3

Irritation parameter:

cornea opacity score

Remarks:

Opacity

Basis:

other: Test

Time point:

other: Maximal score at 240 minutes

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 4

Irritation parameter:

cornea opacity score

Remarks:

Opacity

Basis:

other: Control

Time point:

other: Maximal score at 240 minutes

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 5

Irritation parameter:

other: Fluorescein uptake

Basis:

other: Test

Time point:

other: Maximal score at 240 minutes

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Results 6

Irritation parameter:

other: Flourescein uptake

Basis:

other: Control

Time point:

other: Maximal score at 240 minutes

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

Corneal Opacity
Individual scores for corneal opacity are given in Table 1.
No corneal effects were noted in the test eyes or control eyes during the study period.

Corneal Thickness
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 comparable to that observed in the control eyes over the same period.

Corneal Condition
The condition of the corneal epithelium following treatment is given in Table 4.
The condition of the corneal epithelium of the test eyes and control eyes appeared normal during the study period.

Fluorescein Uptake
Individual scores for fluorescein uptake are given in Table 5.
No fluorescein uptake was noted in the test eyes or control eyes 240 minutes after treatment.

Any other information on results incl. tables

Interpretation of Results

The data for all endpoints was assessed and an estimate of the test item 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:

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.

Applicant's summary and conclusion

Interpretation of results:

other: the test item was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Following assessment of the data for all endpoints, the test item was considered unlikely 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 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. 

Methods. 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°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
Cldyx Area	Intx Area	60mins	120 mins	240 mins	60 mins	120 mins	240 mins
0	0	10.1	7.5	5.3	9.2	6.4	3.9	normal

Conclusion. Following assessment of the data for all endpoints the test item was considered unlikely to have the potential to cause severe ocular irritancyin 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