Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2008 to 16 December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
dark red powder
Batch Number MB-1-A
Storage: room temperature in dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): free access to 2014 Teklad Global Rodent diet
- Water (e.g. ad libitum): free access to mains tap water
- Acclimation period:at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): Approx. 15
- Photoperiod (hrs dark / hrs light): 12 dark: 12 light

Study design: in vivo (non-LLNA)

Induction
Concentration / amount:
Not applicable
Challenge
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable

Study design: in vivo (LLNA)

Vehicle:
other: ethanol/distilled wtaer 7:3
Concentration:
25%, 10% or 5% in ethanol/distilled water 7:3
No. of animals per dose:
4 mice per dose level
Details on study design:
Proliferation response of lymph node cells expressed as the number of radioactive disintegrations per minute per lymph node.
RANGE FINDING TESTS:
None conducted


MAIN STUDY
- 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 node (disintegrations per minute/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".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in ethanol/distilled water 7:3. Information available 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 ul 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.
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 pl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 uCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 uCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No information available

Results and discussion

Positive control results:
A group of five animals was treated with 50 ul (25 ul per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in ethanol/distilled water 7:3 at a concentration of 15% v/v. A further control group of five animals was treated with ethanol/distilled water 7:3 alone.
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 ethanol/distilled water 7:3: 15%
Stimulation Index: 9.49
Result: Positive
alpha-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See Table 1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 1

Any other information on results incl. tables

Table 1. Disintegrations per minute, disintegrations per minute/node and stimulation index

 Concentration (% w/w) in ethanol/distilled water 7:3  dpm  dpm/Node  Stimulation Index (3.0 or greater = positive result)  Result
 Vehicle  6210.17  776.27  na  na
 5  8090.23  1011.28  1.30  negative
 10  6696.62  837.08  1.08  negative
 25  5642.15  705.27  0.91  negative

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.
Executive summary:

The test material, INK BH11 M, was evaluated for its sensitising potential in mice in a Local Lymph Node Assay. The results obtained under the experimental conditions employed demonstrated that the test material did not provoke any reaction of cutaneous sensitization in the animals examined. No abnormalities were noted.The test material was considered to be a non-sensitiser under the conditions of the test.