Condensation products of oil shale alkylresorcinols and formaldehyde

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 to 05 April 2004.

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:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

-

EC Number:

447-830-3

EC Name:

-

Molecular formula:

Not applicable to this UVCB substance.

IUPAC Name:

2-ethylphenol; 3,5-dimethylphenol; formaldehyde; phenol

Test material form:

other: solid resin

Details on test material:

Date received: 30 January 2004
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK.
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): free access to certified rat and mouse feed
- Water (e.g. ad libitum): free access to mains tap water
- Acclimation period: at least 5 days
- Other: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

IN-LIFE DATES: From: 24 March 2004 To: 05 April 2004

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5%, 10% or 25% w/w in acetone/olive oil 4:1.

No. of animals per dose:

4 females per dose

Details on study design:

RANGE FINDING TESTS:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test material at a concentration of 25 % w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

MAIN STUDY
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 ADMISISTRATION
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 ³H-methyl thymidine (³HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 μCi 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 were recorded.

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 190 g) for ten minutes. 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 β-scintillation counting. The "Poly QTM" 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.

INTERPRETATION OF RESULTS
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of ³HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ³HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in ³HTdR incorporation, will be classified as a "non-sensitiser".

Positive control substance(s):

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

Statistics:

none

Results and discussion

Positive control results:

Sensitising. See table 2.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle: N/A 5 % in vehicle: 3.55 10 % in vehicle: 6.87 25 % in vehicle: 14.75

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle: 6937.51 5 % in vehicle: 24627.15 10 % in vehicle: 47678.18 25 % in vehicle: 102309.20

Any other information on results incl. tables

Preliminary Screening Test

There were no signs of systemic toxicity noted.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 1.

A stimulation index of greater than 3 was recorded for the three concentrations of the test material (5%, 10% and 25% w/w in acetone/olive oil 4:1).

Clinical Observations, Bodyweights and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. Brown-coloured staining was noted on the ears of all animals treated with the test material at a concentration of 25%. Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1: Dpm, Dpm/Node and Stimulation Index (SI)

Concentration (% w/w) in acetone/olive oil 4:1	Dpm	Dpm/Node^a	Stimulation Index (SI)^b	Result
Vehicle	6937.51	867.19	N/A	N/A
5	24627.15	3078.39	3.55	Positive
10	47678.18	5959.77	6.87	Positive
25	102309.20	12788.65	14.75	Positive

a = Dpm/node obtained by dividing the Dpm value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

Table 2: Stimulation Index for Positive Control

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index (SI)^b	Result
5	1.76	Negative
10	2.78	Negative
25	5.06	Positive

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was performed in accordance with the OECD 429 guideline and to GLP standard in a certified laboratory.

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 % or 25 % w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Positive results were observed at all concentrations The test material was therefore considered to be a sensitiser under the conditions of the test.