Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted GLP compliant study to recognised internaional test methods

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:
- Name of test material (as cited in study report): THPI D4 (1,2,3,6 tetra-hydro-phthalimide)
- Physical state: Solid
- Analytical purity: 98.26%
- Lot/batch No.: STI4209019
- Expiration date of the lot/batch: 2010-01-19
- Storage condition of test material: Room temperature, protectd from light and humidity

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories BV
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19 - 22 g
- Housing:Group caged in Makrolon Type II cages
- Diet (e.g. ad libitum): Harlan pelleted standard diet, ad libitum
- Water (e.g. ad libitum): municipal supply drinking water, ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 45 - 65%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark


IN-LIFE DATES: From: 2009-03-10 To: 2009-04-01

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 10 and 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Up to 25% in dimethylformamide
- Irritation: None observed following 3 consecutive days application at 10 and 25% concentration
- Lymph node proliferation response: Not assessed


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Random allocation to 4 treatment groups to give a total of 4 groups, each of 5 animals
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Minimum of a 3 fold increase in Stimulatioin Index relative to controls


TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was dissolved in dimethylformamide at the highest achieveable concentration of 25%. Lower concentrations of 10 and 5% were prepared by serial dilution.

Animals of each test group treated by topical application to the dorsal surface of both ears once daily for 3 consecutive days. A volume of 25 μL applied on each occasion. Control animals treated with the vehicle alone.

Five days following the first topical application all animals were treated by intravenous injection of 250 μL 3H-methyl thymidine (activity 19.9 μCi / animal). Five hours later animals were killed and draining lymph modes removed and pooled per animal. Cell suspensions were prepared, incubated to precipitate macromolecules. The precipitates were re-suspended in 5% trichloroacetic acid, mixed with a scintillation liquid and the level of radiolabelled thymidine incorporation determined by β-scintillation counting, recorded as the number of radioactive disintegrations / minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
ANOVA (Dunnett test) undertaken to examine differences between treatment and control groups

Results and discussion

Positive control results:
EC3 value (estimated concentration to give a Stimulation Index of 3) = 7.8% of α-cinnamicaldehyde

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle control - 1.00 5% test substance - 1.00 10% test substance - 0.78 25% test substance - 1.07
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Vehicle control - 457.6 5% test substance - 456.4 10% test substance - 358.9 25% test substance - 490.8 Cited values represent the sum of measured values from all lymph nodes within a group divided by the number of lymph nodes assayed frpom the group

Any other information on results incl. tables

All treated animals survived the study period and no signs of toxicity were observed.

Stimulation indices of 1.00, 0.78 and 1.07 were determined with the test substance at concentrations of 5, 10 and 25%.

The EC3 value could not be determined as no tested concentration gave a Stimulation Index greater than 3

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test substance is not a skin sensitiser under the conditions of the Local Lymph Node Assay
Executive summary:

The test substance is not a skin sensitiser under the conditions of the Local Lymph Node Assay