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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordane with internationally recognised test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisobutyl hexahydrophthalate
EC Number:
275-069-5
EC Name:
Diisobutyl hexahydrophthalate
Cas Number:
70969-58-3
Molecular formula:
C16H28O4
IUPAC Name:
bis(2-methylpropyl) cyclohexane-1,2-dicarboxylate
Details on test material:
- Name of test material : DIBE (diisobutylhexahydrophthalate)
- Physical state: Liquid
- Lot/batch No.: T60211/349
- Expiration date of the lot/batch: 2012-12-15
- Storage condition of test material: room temperature, protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A.
- Age at study initiation: 7 - 9 weeks
- Weight at study initiation: 19 21 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): $RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-08-02 To: 2012-08-17

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Very slight irritation observed at 100% concentration. No irritation observed at lower concentrations of 5 - 50%. Ear thickness measurement was acceptable (less than 25% increase) at 5 and 10% concentrations. Biopsy (ear punch) weight acceptable (less than 25% increase) at 5 - 50% concentrations.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Results and discussion

Positive control results:
SI = 5.74

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Control - 1.00 Low dose (10%) - 2.26 Mid dose (25%) - 2.32 High dose (50%) - 3.49
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: BrdU labelling index/group Control - 0.151 +/- 0.039 Low dose (10%) - 0.340 +/- 0.137 Mid dose (25%) - 0.350 +/- 0.053 High dose (50%) - 0.526 +/- 0.088

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The calculated stimulation indices (SI) were 2.26, 2.32 and 3.49, respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice
Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442b - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in a dose-related and statistically significant increases in cell proliferation of draining lymph nodes in all treated groups when compared to controls. The calculated stimulation indices (SI) were 2.26, 2.32 and 3.49, respectively at low, mid- and high dose levels. Dose-related and statistically significant increases in cell proliferation of draining lymph nodes were observed in all treated groups when compared to controls. The calculated stimulation indices (SI) were 2.26, 2.32 and 3.49, respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice

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