Tar acids (shale oil), C6-9-fraction, alkylphenols

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March 2004 - 07 April 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5-methylresorcinol

EC Number:

207-984-2

EC Name:

5-methylresorcinol

Cas Number:

504-15-4

Molecular formula:

C7H8O2

IUPAC Name:

5-methylbenzene-1,3-diol

Constituent 2

Reference substance name:

Resorcinol

EC Number:

203-585-2

EC Name:

Resorcinol

Cas Number:

108-46-3

Molecular formula:

C6H6O2

IUPAC Name:

benzene-1,3-diol

Constituent 3

Reference substance name:

2,5-dimethylresorcinol

EC Number:

207-688-3

EC Name:

2,5-dimethylresorcinol

Cas Number:

488-87-9

Molecular formula:

C8H10O2

IUPAC Name:

2,5-dimethylbenzene-1,3-diol

Constituent 4

Reference substance name:

2-methylresorcinol

EC Number:

210-155-8

EC Name:

2-methylresorcinol

Cas Number:

608-25-3

Molecular formula:

C7H8O2

IUPAC Name:

2-methylbenzene-1,3-diol

Constituent 5

Reference substance name:

4-methylresorcinol

EC Number:

207-827-8

EC Name:

4-methylresorcinol

Cas Number:

496-73-1

Molecular formula:

C7H8O2

IUPAC Name:

4-methylbenzene-1,3-diol

Constituent 6

Reference substance name:

4,5-dimethylbenzene-1,3-diol

EC Number:

610-895-5

Cas Number:

527-55-9

Molecular formula:

C8H10O2

IUPAC Name:

4,5-dimethylbenzene-1,3-diol

Constituent 7

Reference substance name:

5-ethylbenzene-1,3-diol

Cas Number:

4299-72-3

Molecular formula:

C8H10O2

IUPAC Name:

5-ethylbenzene-1,3-diol

Constituent 8

Reference substance name:

2-ethyl-5-methylbenzene-1,3-diol

Cas Number:

27465-63-0

Molecular formula:

C9H12O2

IUPAC Name:

2-ethyl-5-methylbenzene-1,3-diol

Constituent 9

Reference substance name:

5-ethyl-2-methylbenzene-1,3-diol

Cas Number:

34745-52-3

Molecular formula:

C9H12O2

IUPAC Name:

5-ethyl-2-methylbenzene-1,3-diol

Constituent 10

Reference substance name:

2,4,5-trimethylbenzene-1,3-diol

Cas Number:

20427-81-0

Molecular formula:

C9H12O2

IUPAC Name:

2,4,5-trimethylbenzene-1,3-diol

Constituent 11

Reference substance name:

2,4-dimethylresorcinol

EC Number:

211-213-5

EC Name:

2,4-dimethylresorcinol

Cas Number:

634-65-1

Molecular formula:

C8H10O2

IUPAC Name:

2,4-dimethylbenzene-1,3-diol

Constituent 12

Reference substance name:

Phenol

EC Number:

203-632-7

EC Name:

Phenol

Cas Number:

108-95-2

Molecular formula:

C6H6O

IUPAC Name:

phenol

Constituent 13

Reference substance name:

Cresol

EC Number:

215-293-2

EC Name:

Cresol

Cas Number:

1319-77-3

Molecular formula:

C7H8O

IUPAC Name:

cresol

Constituent 14

Reference substance name:

not applicable

Molecular formula:

not applicable

IUPAC Name:

not applicable

Test material form:

liquid

Details on test material:

Sponsor's identification: Total shale oil phenols
Description: dark brown extremely viscous liquiy
Batch number: Part No. 3
Date received: 30 January 2004
Storage conditions: room temperature, in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaBkI) strain mice were supplied by B & K Universal Ltd, Hull, UK. On
receipt the animals were randomly allocated to cages. The animals were nulliparous and
non-pregnant. After an acclimatisation period of at least five days the animals were selected at
random and given a number unique within the study by indelible ink-marking on the tail and a
number written on a cage card. At the start of the study the animals were in the weight range of
15 to 23 g, and were eight to twelve weeks old.

The animals were individually housed in suspended solid-floor polypropylene cages furnished
with softwood woodflakes. Free access to mains tap water and food (Certified Rat and Mouse
Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK) was allowed throughout the
study.

The temperature and relative humidity were controlled to remain within target ranges of 19 to
25°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
approximately 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.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5 %, 10 % and 25 % (w/w) in acetone/olive oil

No. of animals per dose:

4

Details on study design:

Test Material Administration

Groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% w/w
in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not
produce systemic toxicity or excessive local irritation at the highest suitable concentration. The
mice were treated by daily application of 25 µl of the appropriate concentration of the test
material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test
material formulation was administered using an automatic micropipette and spread over the dorsal
surface of the ear using the tip of the pipette.

A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration

Five days following the first topical application of the test material (Day 6) all mice were injected
via the tail vein with 250 µl of phosphate buffered saline containing *H-methyl thymidine
CHTAR: 80 wCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total
of 20 Ci to each mouse.

Observations

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily
basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the study wererecorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures

Termination: Five hours following the administration of 3HTdR all mice were killed by carbon
dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and
pooled for each experimental group. For each group 1 ml of phosphate buffered saline (PBS) was
added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells
was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The
lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with
the project number and dose concentration. The lymph node cell suspension was transferred to a
10 ml centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all
remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node
cells were pelleted at 1400 rpm (approximately 190g) for tenminutes. The pellet was
resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the
pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After overnight incubation at 4°C, the precipitates
were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended
in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase ‘Trisafe’). 3HTdR
incorporation was measured by B-scintillation counting. The "Poly Q™" vials containing the
samples and scintillation fluid were placed in the sample changer of the scintillator and left for
approximately twenty minutes. The purpose of this period of time in darkness was to reduce the
risk of luminescence, which has been shown to affect the reliability of the results. After
approximately twenty minutes, the vials were shaken vigorously. The number of radioactive
disintegrations per minute was then measured using the Beckman LS6500 scintillation system
(Beckman Instruments Inc, Fullerton, CA).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of alpha-HEXYLCINNAMALDEHYDE as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% and 25% w/w. A further control group of four animals was treated with acetone/olive oil 4:1
alone.

Results. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% (w/w)) SI Index result
5 1.76 negative
10 2.78 negative
25 5.06 postivie

Conclusion: alpha-HEXYLCINNAMALDEHYDE was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 2.38

Test group / Remarks:

5 % (w/w) in acetone/olive oil 4:1

Results 2

Key result

Parameter:

SI

Value:

ca. 5.42

Test group / Remarks:

10 % (w/w) in acetone/olive oil 4:1

Results 3

Key result

Parameter:

SI

Value:

ca. 10.47

Test group / Remarks:

25 % (w/w) in acetone/olive oil 4:1

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test materialin the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

The method was designed to meet the requirements of the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay” (adopted 24 April 2002)

Methods: Following a preliminary screening test, three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% and 25% w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results: The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index (SI)	Result
5	2.38	Negative
10	5.52	Positive
25	10.47	Postive

Conclusion: The test material was considered to be a sensitiser under the conditions of the test.