Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines

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)
Version / remarks:
Reliability scoring based on 2002 guideline
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Gluconic Acid Technical 50/50
- Physical state: liquid
- Analytical purity: 54.4%
- Lot/batch No.: S293E
- Expiration date of the lot/batch: 01-Aug-2014
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid-floor polypropylene cages
- Diet (e.g. ad libitum): 2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days


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


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: not applicable (see LLNA below)
Vehicle:
other: not applicable (see LLNA below)
Concentration / amount:
not applicable (see LLNA below)
Challengeopen allclose all
Route:
other: not applicable (see LLNA below)
Vehicle:
other: not applicable (see LLNA below)
Concentration / amount:
not applicable (see LLNA below)
No. of animals per dose:
not applicable (see LLNA below)
Details on study design:
not applicable (see LLNA below)
Challenge controls:
not applicable (see LLNA below)

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Groups of four mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in dimethyl formamide.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Not reported
- Irritation: Not irritating
- Lymph node proliferation response: Not reported


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: 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 3H-methyl thymidine (3HTdR) incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated daily with an application of 25 microlitres of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days. 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 four mice received the vehicle alone in the same manner. Five days following the first topical application of the test material all mice were injected via the tail vein with 250 microlitres of phosphate buffered saline containing 3HTdR. Five hours following the administration of 3HTdR all mice were killed. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% trichloroacetic acid (TCA). After approximately 18 hours incubation at approximately 4 degrees Celsius, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3HTdR was counted by beta-scintillation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable

Results and discussion

Positive control results:
The positive control test resulted in a stimulation index of 4.24, therefore, a positive sensitisation result.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle: not applicable Test concentration 25%: 1.10 Test concentration 50%: 1.00 Test concentration 100%: 0.81
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 6767.65 Test concentration 25%: 7471.43 Test concentration 50%: 6767.38 Test concentration 100%: 5494.77

Any other information on results incl. tables

There were no deaths and no signs of systemic toxicity reported. Body weight changes between Day 1 and Day 6 were similar among test and control animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information