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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:
20 Sep - 4 Oct 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
study performed before release of OECD Guideline 429
GLP compliance:
yes (incl. QA statement)
Remarks:
Federal Office of Public Health, the Swiss Agency for the Environment, Forests and Landscape and the Intercantonal Office for the Control of Medicines, Bern, Switzerland
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-cyclohexylethyl (E)-but-2-enoate
EC Number:
944-449-9
Molecular formula:
C12H20O2
IUPAC Name:
1-cyclohexylethyl (E)-but-2-enoate

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 10 weeks old
- Weight at study initiation: 17.0 - 19.9 g
- Housing: in groups of 4 in Makrolon cages (until intravenous injection of 3H-methyl thymidine: type-3 cages; afterwards until necropsy: type-1 cages) on standard softwood bedding
- Diet: pelleted standard Kliba 3433, mouse maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: approx. 7 days

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

Study design: in vivo (LLNA)

Vehicle:
other: acetone
Concentration:
1, 5 and 25% (w/w)
No. of animals per dose:
4
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A test substance is regarded as a sensitizer if two criteria are fulfilled. First, the exposure to at least one concentration of the test substance resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control animals, as indicated by the Stimulation Index (SI). Second, the data must be 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: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance at concentrations of 1, 5 and 25% or the vehicle acetone. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied. The application was repeated on Days 2 and 3. Five days after the first topical application, each experimental mouse was administered 250 µl of 80.56 µCi/mL 3H-methyl thymidine (3H-TdR) by intravenous injection via the tail vein. Approximately five hours later, the draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions of pooled lymph node cells were prepared in PBS by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). Pooled lymph node cells were washed three times with PBS, resuspended in 5% trichloroacetic acid and incubated at approx. 4 °C overnight for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloracetic acid (1 mL). The level of 3H-TdR incorporation was the measured on a ß-scintillation counter.
Positive control substance(s):
other: 2,4-Dinitrochlorobenzene (DNCB)

Results and discussion

Positive control results:
The results of a reliability test with three concentrations of DNCB in acetone/olive oil (4:1 v/v) was performed not more than 6 months previously (26 Apr - 16 May 2000) in CBA/Jlbm (SPF) mice using the same materials and essential procedures. The SI values calculated for the substance concentrations 0.01, 0.1 and 0.5% were 1.8, 6.4 and 12.2 respectively.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
1% test substance
Key result
Parameter:
SI
Value:
2.9
Test group / Remarks:
5% test substance
Key result
Parameter:
SI
Value:
6.7
Test group / Remarks:
25% test substance
Cellular proliferation data / Observations:
EC3 CALCULATION: No EC3 value was calculated in the study report.

VIABILITY/MORTALITY: No deaths occurred during the study period.

CLINICAL OBSERVATIONS: No symptoms of Iocal toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS: 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.

Any other information on results incl. tables

Table 1. DPM counts and SI values

 

Body weight (g)

DPM count

DPM – BG*

DPM per lymph node

SI value

Day 1

Day 6

BG I

-

-

1

-

-

-

BG II

-

-

0

-

-

-

Vehicle Control Group

19.2 ± 0.8

20.3 ± 0.9

1746

1745

218

-

Test Group I - 1%

17.9 ± 0.4

19.0 ± 0.6

1630

1629

204

0.9

Test Group II - 5%

17.8 ± 0.7

19.0 ± 0.7

5077

5076

635

2.9

Test Group III - 25%

18.4 ± 0.4

19.8 ± 0.4

11660

11659

1457

6.7

BG: background (1 mL 5% trichloroacetic acid) in duplicate

*: The mean value was taken from the figures BG I and II

**: Since the Iymph nodes of the animals of a dose group were pooled, DPM/node was determined while the measured value was divided by the number of lymph nodes (8) pooled.

Applicant's summary and conclusion

Interpretation of results:
other: Skin Sens. Cat. 1B
Remarks:
according to Regulation (EC) 1272/2008
Conclusions:
Under the conditions of the mouse Local Lymph Node Assay (LLNA) the test substance revealed sensitising properties.
Executive summary:

The skin sensitisation potential of the test substance was determined by a LLNA test according to OECD Guideline 429 in compliance with GLP (2000). After treatment of CBA/CaOlaHsd mice with the test substance, the test substance produced stimulation indices of 0.9, 2.9 and 6.7 at test substance concentrations of 1, 5 and 25% in acetone, respectively. The EC3 value was considered to be 5.5% (not given in study report).