Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-04-22 to 2008-05-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Sponsor and 902/695838/D/2/1

RADIOLABELLING INFORMATION
- Specific activity: 2.0 Ci/mmol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4°C in the dark
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in the vehicle at desired dosing concentration

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in dimethyl formamide

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dissolved in dimethyl formamide at 50 and 25% w/w test ingredient to vehicle for preliminary screening
Dissolved in dimethyl fornamide at 25, 10, and 5% w/w test ingredient to vehicle for the main test

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Females nulliparous and non-pregnant:yes
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: Suspended solid-floor polypropylene cages furnished with softwood woodflake
- Diet (e.g. ad libitum): Certified Rat and Mouse Diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days

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

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
50 and 25% w/w test ingredient to vehicle for preliminary screening
25, 10, and 5% w/w test ingredient to vehicle for the main test
No. of animals per dose:
1 female for the preliminary study
5 females for the main test (including vehicle control)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Compound was completely soluble to dosing level necessary (50% w/w test ingredient to vehicle)
- Systemic toxicity: The animal treated with the test material at a concentration of 50% w/w in dimethyl formamide was humanely killed, pre-dose on Day 3, due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted were hunched posture, lethargy and ptosis.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- 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 from each individual animal 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 3 HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3 HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the test material at concentrations of 5%, 10% or 25% w/w in dimethyl foimamide. 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 .il 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 five 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 tl of phosphate buffered saline (PBS) containing 3 H-methyl thymidine (3 HTdR:80uCiiml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 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 (prior to dosing) and Day 6 (prior to termination).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

Positive control 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 dimethyl formamide Stimulation Index Result
5 2.05 Negative
10 3.52 Positive
25 6.87 Positive

Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in dimethyl formamide.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 0
Test group / Remarks:
Test Group 1 - 5 females - negative control
Key result
Parameter:
SI
Value:
ca. 16.9
Variability:
Significantly different from control group p<0.01
Test group / Remarks:
Test Group 2 - 5 females - 5% w/w test ingredient in dimethyl fornamide
Key result
Parameter:
SI
Value:
ca. 20.3
Variability:
Significantly different from control group p<0.01
Test group / Remarks:
Test Group 3 - 5 females - 10% w/w test ingredient in dimethyl fornamide
Key result
Parameter:
SI
Value:
ca. 24.14
Variability:
Significantly different from control group p<0.001
Test group / Remarks:
Test Group 4 - 5 females - 25% w/w test ingredient in dimethyl fornamide
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal 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 3 HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3 HTdR incorporation will be
classified as a "non-sensitiser".

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

BODY WEIGHTS 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.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test material was classified as a contact sensitiser (Category 1) according to the GHS criteria.