Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 March 2004 to 18 March 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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 2 December 2002 Date of Signature: 13 February 2003
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TIPX
- Substance type: Colourless liquid.
- Physical state: Liquid.
- Lot/batch No.: 042028.
- Storage condition of test material: Approximately 4ºC in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaBkl)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: B&K Universal Ltd, Hull, United Kingdom.
- Age at study initiation: 8 to 12 weeks old.
- Weight at study initiation: 15 to 23 g.
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (Certified Rat and Mouse Diet (Code 5LF2), supplied by BCM IPS Limited, London, United Kingdom) ad libitum throughout the study.
- Water: Mains tap water ad libitum throughout the study.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25ºC.
- Humidity (%): 30 to 70%.
- Air changes (per hr): Approximately 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.

IN-LIFE DATES: From: Day 0 To: Day 6.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25% , 50% or 100%.
No. of animals per dose:
4 animals at each dose and the control group.
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 undiluted test material to the dorsal surface of each ear for 3 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 was recorded on Day 1 (prior to dosing) and on Day 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse.
- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes 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".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was used undiluted and freshly prepared in acetone/olive oil 4:1.
(MAIN TEST)
Groups of four mice were treated with the undiluted test material or the test material at concentrations of 25% or 50% v/v in the vehicle. 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:
The 3H-Methyl Thymidine was administered on Day 6. All mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) 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.
- Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.
- Preparation of Single Cell Suspension
A single cell suspension of the 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 cells suspension was transferred to a 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 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 approximately eighteen hours incubation at approximately 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 beta 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 (Beckman Instruments Inc, Fullerton, CA).
Positive control substance(s):
other: alpha-hexylcinnamaldehyde at 5, 10, 25% v/v in acetone/olive oil 4:1
Statistics:
Not reported.

Results and discussion

Positive control results:
Not applicable.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation index is given in the table below.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per lymph node is given in the table below.

Any other information on results incl. tables

Table DPM, DPM/Node and Stimulation Index (SI)

Concentration (% v/v) in acetone/olive oil 4:1 Dpm Dpm / Nodea Stimulation Indexb
(SI)
Result
Vehicle 3503.62 437.95 N/A N/A
25 25637.13 3204.64 7.3 Positive
50 46794.79 5849.35 13.4 Positive
100 68954.25 8619.28 19.7 Positive

dpm= Disintegrations per minute

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

Other effects

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test. The test material is classified as R43 May cause sensitisation by skin contact under Council Directive 67/548/EEC and Category 1 H317: May cause an allergic skin reaction under under Regulation (EC) No 1272/2008.
Executive summary:

Introduction. 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 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 undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 25% or 50% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results. The Stimulation Index 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 (% v/v) in acetone/olive oil 4:1

Stimulation Index

Result

25

7.3

Positive

50

13.4

Positive

100

19.7

Positive

Conclusion. The test material was considered to be a sensitiser under the conditions of the test. The test material is classified as R43 May cause sensitisation by skin contact under Council Directive 67/548/EEC and Category 1 H317: May cause an allergic skin reaction under under Regulation (EC) No 1272/2008.