Petroleum, synthetic, hydrodesulfurized full-range coker

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 01 August 2012 and 25 September 2012

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.29 to 2.53 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Certified Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

A quantity of 0.5 ml of the test item was applied directly to the skin

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

3

Details on study design:

On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5 ml of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.
Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in 74% Industrial Methylated Spirits.

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal: 72323 male

Time point:

other: Highest score at 1, 24, 48, 72 hours and 7 days

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Brown coloured staining and loss of skin elasticity noted

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal: 72324 Male

Time point:

other: Highest score at 1, 24, 48, 72 hours and 7 days

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Brown coloured staining, loss of skin elasticity and slight desquamation noted

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal: 72454 male

Time point:

other: Highest score at 1, 24, 48, 72 hours and 7 days

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Brown coloured staining and loss of skin elasticity noted

Irritation parameter:

other: Oedema Formation

Basis:

animal: 72323 male

Time point:

other: Highest score at 1, 24, 48, 72 hours and 7 days

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

other: Oedema Formation

Basis:

animal: 72324 male

Time point:

other: Highest score at 1, 24, 48, 72 hours and 7 days

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

other: Oedema Formation

Basis:

animal: 72454 male

Time point:

other: Highest score at 1, 24, 48, 72 hours and 7 days

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

The individual scores for erythema/eschar and oedema are given in Table 1.
Brown coloured staining, not preventing evaluation of skin responses, was noted at all treated skin sites during the study.
Very slight erythema, with or without very slight oedema, was noted at all treated skin sites immediately and one hour after patch removal. Very slight to well defined erythema and slight oedema were noted at all treated skin sites at the 24 Hour observation with very slight to well-defined erythema and very slight to slight oedema at the 48 Hour observation. Well defined erythema and very slight oedema were noted at two treated skin sites with very slight erythema noted at one treated skin site at the 72 Hour observation.
Loss of skin elasticity was noted at one treated skin site at the 48 Hour observation and at all treated skin sites at the 72 Hour observation.
Slight desquamation was noted at one treated skin site at the 7 Day observation and the other two treated skin sites appeared normal at this time.

Other effects:

Bodyweight
Individual bodyweights and bodyweight changes are given in Table 2.
All animals showed expected gain in bodyweight during the study.

Interpretation of Results

Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme

The scores for erythema and oedema at the 24 and 72‑Hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test item. The test item was classified according to the following scheme devised by Draize J H (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the,,, p.46‑59:

Primary Irritation Index	Classification of Irritancy
0	Non-irritant
> 0 to 2	Mild irritant
> 2 to 5	Moderate irritant
> 5 to 8	Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.

The results were also interpreted according toRegulation (EC) No 1272/2008, relating to the Classification, Packaging and Labelling of Dangerous Substances.

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: Draize classification scheme

Conclusions:

The test item produced a primary irritation index of 3.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
The test item did not meet the criteria for classification as irritant or corrosive according to Regulation (EC) No 1272/2008, relating to the Classification, Packaging and Labelling of Dangerous Substances.

Executive summary:

Introduction. The study was performed to assess the irritancy potential of the test item to the skin of the New Zealand White rabbit. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 24 April 2002)

Method B4 Acute Toxicity (Skin Irritation) of Commisssion Regulation (EC) No. 440/2008

Results. A single 4-Hour, semi-occluded application of the test item to the intact skin of three rabbits produced very slight to well-defined erythema, slight oedema and loss of skin elasticity. Slight desquamation was noted at one treated skin site at the 7 -Day observation and the other two treated skin sites appeared normal at this time.

Conclusion. The test item produced a primary irritation index o f3.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

The test item did not meet the criteria for classification as irritant or corrosive according to Regulation (EC) No 1272/2008, relating to the Classification, Packaging and Labelling of Dangerous Substances.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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.

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)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Not applicable

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.

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

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

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

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

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

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.

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.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The test item did not meet the criteria for classification as skin or eye irritant or corrosive according to CLP.