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Toxicological information

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:
25.09. - 18.12.2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted June 24, 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 Netherlands B.V. Postbus 6174 NL - 5960 AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks (beginning of acclimatization period)
- Weight at study initiation: 16.0 g - 22.3 g (beginning of acclimatization period)
- Housing: in groups of four in Makrolon type-3 cages
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 -15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: 25.09. - 09.10.2002

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 1, 2.5, 5, 10 and 25 % (w/v)
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in 2 mice with concentrations of 1 %, 5 %, 10 % and 25 % (w/v).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.

Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1 %, 2.5 %, 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were administered with 250 µL of 83.64 µCi/mL 3HTdR (equal to 20.9 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing three times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C overnight for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a p-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of body weight were calculated.

Results and discussion

Positive control results:
The positive control substance alpha-hexylcinnamaldehyde was found to be a sensitizer and an EC3 value of 11.3 % (w/v) was derived with stimulation indices of 2.6 and 7.1 at test item concentrations of 10 % and 25 % (w/v), respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: group 2 (1 % (w/v)): 1.7 group 3 (2.5 % (w/v)): 1.4 group 4 (5 % (w/v)): 1.3 group 5 (10 % (w/v)): 1.3 group 6 (25 % (w/v)): 1.0 No dose response relation was observed
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: group 1 (control): 5518 dpm group 2 (1 % (w/v)): 9521 dpm group 3 (2.5 % (w/v)): 7969 dpm group 4 (5 % (w/v)): 7182 dpm group 5 (10 % (w/v)): 7090 dpm group 6 (25 % (w/v)): 5490 dpm

Any other information on results incl. tables

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information