Registration Dossier

Diss Factsheets

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Sep 2005 to 17 Oct 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 429)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Relative humidity exceeded OECD 429 recommended level of 70%
Principles of method if other than guideline:
Relative humidity exceeded OECD 429 recommended level of 70%
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
- Name of test material (as cited in study report: DESMA
- Physical state: Yellowish viscous liquid
- Analytical purity: >98%
- Lot/batch no.: FC 02/139
- Expiration date of the lot/batch: 31 Dec 2005
- Storage condition of test material: Room temperature, protected from light and moisture

In vivo test system

Test animals

other: CBA/CaOlaHsd
Details on test animals and environmental conditions:
- Source: Harlan Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.8 g - 20.3 g
- Housing: Single caging in Makrolon Type I, with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet (Harlan Winkelmann GmbH) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days; animals were examined for health prior to inclusion on the study
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 96%
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
IN-LIFE DATES: From: 14-Sep-2005 To: 21-Sep-2005

Study design: in vivo (LLNA)

4, 8, or 16% (w/v)
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS: 2, 4, 8, and 16% (w/v) DESMA in dimethylformamide (DMF) was applied once to one female mouse ear per concentration (two mice total).
- Compound solubility: Soluble in dimethylformamide (DMF) following stirring at up to at least 16%
- Irritation: No irritation observed

- Name of test method: 3H-methyl thymidine (3HTdR) administration followed by beta-scintillation counter analysis of disintegrations per minute.
- Criteria used to consider a positive response: 1) A 3-fold or greater increase in incorporation of 3HTdR in the lymph node relative to the vehicle control with evidence of a dose-response relationship is considered indicative of a positive response. 2) The data must be compatible with a conventional dose response, although allowance must be made (especially at high concentrations) for either local toxicity or immunological suppression.
Mice (4 females/group) received topical application of 25 uL of 4, 8, or 16% (w/v) DESMA in DMF to the dorsal surface (diameter 8 mm) of each ear lobe once daily for three consecutive days. A vehicle control group of mice (4 females) received DMF using the same method. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of applied test item. Five days after the first application, all mice received 250 uL of 81.5 uCi/mL 3HTdR (corresponds to 20.375 uCi 3HTdR per mouse) via intravenous injection in the tail vein. Five hours after treatment with 3HTdR all animals were euthanized by intraperitoneal Na-thiopental. The draining lymph nodes were excised and pooled (8 lymph nodes per group). Single cell suspensions were prepared by mechanical disaggregation through stainless steel gauze (200 um mesh size). The cells were washed twice with phosphate buffered saline, then resuspended in 5% trichloroacetic acid, and incubated at 4 C for 18 hours to precipitate macromolecules. The precipitates were resuspended in 5% trichloroacetic acid and 3HTdR incorporation was measured in a beta-scintillation counter. Background 3HTdR was assessed in two aliquots of 5% trichloroacetic acid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Mean values and standard deviations were calculated for body weights. The EC3 was calculated by the following equation: EC3 = (a-c)[(3-d)/(b-d)] +c, where (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the stimulation index (S.I) value of 3 on the local lymph node dose response plot. If all S.I. values are less than 3, then the EC3 cannot be calculated.

Results and discussion

Positive control results:
Prior to this study, the positive control was validated in acetone:olive oil (4:1, v/v) in the test system in August 2005.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: The stimulation index (S.I.) was 1.05, 1.03, and 1.36 for each of the 4%, 12%, and 16% test article concentrations, respectively. The EC3 could not be calculated because no test article-treated group achieved an S.I. greater than or equal to 3.
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM per lymph node values were 535.8, 560.5, 553.2, and 729.0 for the vehicle control, 4%, 12%, and 16% test article concentrations, respectively.

Any other information on results incl. tables

All animals survived. No clinical signs of toxicity were observed. Body weights were within the normal range for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results:OECD GHS
Based on the results of this study, CASRN 1101874-33-2 was not sensitizing in the mouse Local Lymph Node Assay.
Executive summary:

OBJECTIVE: The dermal sensitizing potential of DESMA (batch FC 02/139) was evaluated in a local lymph node assay (LLNA) in female CBA mice.

METHODS: The study design was based on OECD Guideline 429 (2002). DESMA was diluted in vehicle (dimethyl formamide (DMF)) to the appropriate concentrations. A pretest determined that 16% DESMA did not cause dermal irritation. Female mice (4/group) received 25 uL of vehicle, 4, 8 or 16% DESMA on three consecutive days by open application on the left and right ears. A hair dryer was used to dry the ear’s surface as quickly as possible to avoid loss of test material. Five days after the first exposure all animals were subjected to a 3H-methyl thymidine treatment and the auricular lymph nodes were removed to measure the amount of proliferative DNA by beta-scintillation counter in disintegrations per minute (DPM). The stimulation index (S.I.) was calculated for each group and, if possible, the concentration at which DESMA could elicit an S.I. greater than or equal to 3 was determined (EC3). Alpha-hexylcinnamic aldehyde (HCA) is tested in this study design every 6 months at the test facility as a positive control and the most recent results indicated the model was valid.

RESULTS: Mean DPM/lymph node values were 535.8, 560.5, 553.2, and 729.0 for the vehicle, 4, 8 and 16% DESMA groups, respectively. The S.I. values for the 4, 8 and 16% dose groups were 1.05, 1.03 and 1.36, respectively. The EC3 could not be calculated because no test article-treated group achieved an SI greater than or equal to 3.

CONCLUSION: Under the conditions of this study, DESMA was not a dermal sensitizer.