Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 04, 2006 - July 05, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2005-11-21
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: BIO 1031/1
Substance type: pure active substance
Physical state: solid
Storage condition of test material: ambient temp., dark, dry, in original container

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mice were supplied by Charles River UK limited. The animals were nulliparous and non-pregnant.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK; ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 per hour
- Photoperiod: 12 hours light/dark cycle
TEST ANIMALS
- Source: Mice were supplied by Charles River UK limited. The animals were nulliparous and non-pregnant.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK; ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 per hour
- Photoperiod: 12 hours light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Animals were treated with the test material at concentrations of 5%, 10% or 25% (w/w) in acetone/olive oil 4:1.
No. of animals per dose:
4 mice in the treatment group and 4 mice served as the control
Details on study design:
RANGE FINDING TESTS: As no toxicological information was available regarding the sysemic toxicity/irritancy potential of the test material a preliminary screening test was performed using 1 mouse. The mouse was treated by daily application of 25µl 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 3 consecutive days. The mouse was observed twice daily an Days 1, 2 and 3 and once on Days 4, 5 and 6. Any signs of toxicity or excessive local irritaion noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 and on Day 6.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25µl of the appropirate concentration of the test material to the dorsal surface of each ear for 3 consecutive days. The teat 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 goup of 4 mice received the vehicle alone in the same manner.

[3H]-METHYL THYMIDINE ADMINSTRATION: 5 days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250µl of phosphate buffered saline (PBS) containing [3H]-methyl thymidine giving a total of 20 µCi to each mouse.

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 test were recorded.

BODYWEIGHTS: The bodyweight of each mouse was recorded on Day 1 and Day 6.

TERMINATION: 5 hours following the administration of [3H]-methyl thymidine all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the 4 mice 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 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 4ml of PBS into a labelled petri dish. The lymph node cell suspension was centrifuged at 1400 rpm for 10 minutes. To precipitate out the radioactive material, the pellet was resuspended in 3ml of 5% trichloroacetic acid (TCA).

DETERMINATION OF THE [3H]-METHYL THYMIDINE INCORPORATION: [3H]-methyl thymidine incorporation was measured by beta-scintillation counting.

INTERPRETATION OF THE RESULTS: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node and as the ratio of [3H]-methyl thymidine incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. 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 [3H]-methyl thymidine incorporation compared to control values.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not applicable

Results and discussion

Positive control results:
5 % (w/w) Stimulation index (SI): 2.50 Result: negative
10 % (w/w) Stimulation index (SI): 4.03 Result: positive
25 % (w/w) Stimulation index (SI): 9.13 Result: positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 5 % (w/w) SI: 0.83 10 % (w/w) SI: 0.83 25 % (w/w) SI: 0.83
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 5 % (w/w) DPM: 10937.99 10 % (w/w) DPM: 10935.69 25 % (w/w) DPM: 10893.52

Any other information on results incl. tables

PRELIMINARY SCREENING TEST: No signs of systemic toxicity were noted. Pale yellow-cloured residual test material was noted on the ear and fur 1 hour post dosing on Day 3. Based on this information the dose levels selected for the main test were 5%, 10% and 25% w/w in vehicle.

MAIN TEST:

CLINICAL OBSERVATIONS AND MORTALITY DATA: There were no deaths. Pale yellow-coloured residual test material was noted on the ears and fur on the animals treated with the test material at a concentration of 25% w/w in vehicle 1 hour post dosing on Days 2 and 3. No signs of systemic toxicity were noted in the test or control groups.

BODYWEIGHT: Bodyweight changes of the test animals between Day 1 and 6 were comparable to those observed in the corresponding control group animals.

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

Table 1: DPM, DPM/node and stimulation index (SI)

Concentration

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

DPM

DPM/nodea

Stimulation index (SI)b

Result

Vehicle

13102.33

1637.79

N/A

N/A

5

10937.99

1367.25

0.83

negative

10

10935.69

1366.96

0.83

negative

25

10893.52

1361.69

0.83

negative

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

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.