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Description of key information

Skin sensitisation: not sensitising (OECD 429; GLP compliant)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-08-17 to 2006-09-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002-04-24
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2005-06-09
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark, desiccated, under nitrogen
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HArlan UK Ltd., Bicester, Oxon, England
- Age at study initiation: Approx. eight to ten weeks of age prior to dosing
- Weight at study initiation: Weight range of 17.0 to 23.0 g
- Housing: The animals were housed individually in polycarbonate cages with woodflake bedding.
- Diet (ad libitum): Standard laboratory rodent diet (Special Diet Services RM1 (E) SQC)
- Water (ad libitum): Drinking water
- Acclimation period: At least five days prior to the start of the study
The mice were also given Nestlets for environmental enrichment.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 2°C
- Relative humidity: 40 to 70%
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethylformamide
Concentration:
10, 25, and 50% w/v of the test item
No. of animals per dose:
5 female mice
Details on study design:
RANGE FINDING TESTS:
The dose levels for the preliminary study were chosen based on the physical properties of the test substance e.g. solubility, viscosity.
Groups of one female were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µl of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette.
The maximum practical concentration for pinna dosing was 50% w/v in DMF. Based on this information the following concentrations were selected for the preliminary study: 10, 25 and 50% w/v.
The mice were killed by carbon dioxide asphyxiation on Day 4 of the study. No further investigations were carried out on these animals.
Results:
There were no deaths during the study. Clinical signs of reaction to treatment comprised:
Group 1 (10% w/v): Wet fur was observed from immediately post-dose on Days 2 and 3, resolving within approximately one hour on each occasion.
Group 2 (25% w/v): Wet fur was observed from immediately post-dose on Day 3, resolving within approximately one hour.
Group 3 (50% w/v): wet fur was observed from immediately post-dose on Day 3, resolving within approximately one hour.
Body weight increases were recorded for all mice over the period of the study.
On the basis of the results from the preliminary study, 50% w/v was considered a suitable high dose level for the main study.

MAIN STUDY
The dosages for the main study were chosen based on the results of the preliminary study. The dosage for the positive control group was chosen based on previous studies conducted at the laboratory performing this study using HCA (hexyl cinnamic aldehyde).
Groups of five mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of each ear using the tip of the pipette. In addition, a group of five mice was similarly dosed with the vehicle alone and a further group of five mice received HCA (hexyl cinnamic aldehyde; positive control) in the same manner.
Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 ml of phosphate buffered saline containing 3H-methyl Thymidine (G E Healthcare UK Limited. specific activity 2.0 Ci/mmol, concentration 1.0 mCi/ml) (3HTdR: 80 mCi/ml) giving a nominal 20 mCi to each mouse.
Five hours following the administration of 3HTdR on Day 6 all mice were killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised for each experimental animal. 1.0 ml of phosphate buffered saline was added to the lymph nodes of each animal. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals:
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding 10 ml phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended in 3 ml trichloroacetic acid. (TCA: 5%) following the final wash.
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 ml 5% TCA and transferred to 10 ml Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative the that recorded for control nodes (test/control ratio).

TREATMENT PREPARATION AND ADMINISTRATION:
A vehicle trial conducted with DIGIRENA SA TEMABF4 anhydrous showed that it formed a clear colourless solution in DMF (dimethylformamide).
The test substance formulations were prepared in DMF at the required concentrations freshly on each day of dosing.
The maximum practical concentration for pinna dosing was 50% w/v in DMF.

OBSERVATIONS:
- Clinical signs: All animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation.
- Body weight: The weight of the mice in the preliminary study were recorded on arrival, Day 1 (first day of dosing) and prior to termination (Day 4).
The weight of each mouse in the main study was recorded on arrival, Day 1(first day of dosing) and prior to termination (Day 6).

INTERPRETATION OF RESULTS:
Results for each treatment group were expressed as the stimulation index. This was obtained by comparing the proliferation in the vehicle treated control group with the values from the three test groups as follows: the ratio of 3HTdR incorporation into lymph node cells, expressed as dpm, relative to that recorded for control lymph nodes is derived for each test group based on the group mean dpm per node. When the stimulation index ≥ 3, the test substance is regarded as a skin sensitizer with a consideration given to dose response and statistical significant (see also "Statistics" below).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Analysis of variance was carried out on the data, from the vehicle control group and the test groups, with treatment group as the factor of interest. If Bartlett's test for homogeneity of variance was significant at the 1% level, the data were logarithmically transformed prior to analysis in order to stabilise the variance (Bartlett, M.S. (1937) Properties of sufficiency and statistical tests. Proceedings of the Royal Society. Series A, 160, 268 - 282 (Also see J.R. Stat. Soc. Suppl 4, 1937, published later)). Comparisons were made between the control and DIGIRENA SA TEMABF4 anhydrous treated groups using Dunnett's test (Dunnett, C.W. (1955) A multiple comparison procedure for comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association,50, 1069 - 1121.)(Dunnett, C.W. (1964) New tables for multiple comparisons with a control. Biometrics, 20, 482 - 491.). To investigate the nature of the dose response curve the linear contrast was isolated.
Analysis of variance was also carried out to compare the positive control group with the vehicle control group.
Positive control results:
Clinical signs observed for the animals receiving the positive control comprised wet fur in all animals from immediately post-dose on Day 1. This sign had resolved in all animals by the pre-dose check on Day 2. Greasy fur was observed from pre-dose on Day 2 and was still present at termination on Day 6. Slight reddening of the skin in the head and cervical region was recorded for all animals from approximately four hours post-dose on Day 2, resolving by the pre-dose check on Day 3. This sign was again observed in all animals from approximately one hour post-dose on day 3 and had resolved by Day 6. In addition underactive behaviour and thin build were observed for one animal on Days 4 and 5, resolving completely by Day 6. A loss in body weight was recorded for one female in the positive control group during the study.
The stimulation index for the positive control substance was 41.2, which proved to demonstrate the reliability and sensitivity of this assay to detect skin sensitization potential in the performing laboratory.
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
10% w/v concentration
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
25% w/v concentration
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
50% w/v concentration
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS:
There were no deaths and no signs of ill health or toxicity were observed in animals receiving the test material or negative control during this study.
No signs of irritation were seen over the dosed area of the animals receiving the test material or negative control during the study.
Wet fur was noted for all negative control and test animals from immediately post dose on Days 1,2 and 3. This sign had resolved in all animals by approximately one hour post-dose on each occasion. White particles on the ears were recorded post-dose on Days 2 and 3 for all animals dosed with the test material at 25% or 50% w/v. This sign had resolved in all animals by the following morning.

BODY WEIGHTS
body weight gain was recorded for three females receiving the negative control and one female receiving the test material at 25 % w/v. All remaining animals gained weight during the study.
Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation

The substance has no skin sensitisation potential based on a reliable local lymph node assay (OECD 429).

Justification for classification or non-classification

Skin sensitisation

The substance has no skin sensitisation potential based on a local lymph node assay (OECD 429). Thus, the substance does not require classification as skin sensitiser according to Regulation (EC) No 1272/2008 and subsequent adaptations.