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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2002 to 02 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-phenoxyethyl isobutyrate
EC Number:
203-127-1
EC Name:
2-phenoxyethyl isobutyrate
Cas Number:
103-60-6
Molecular formula:
C12H16O3
IUPAC Name:
2-phenoxyethyl 2-methylpropanoate
Test material form:
other: liquid (undefined)
Details on test material:
- Name of test material (as cited in study report): Phenoxy ethyl isobutyrate.
- Physical state: Colourless to pale yellow liquid.
- Stability of test material: Stable under storage conditions.
- Storage conditions: At room temperature, in the original container, away from direct sunlight.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 - 12 weeks (beginning of acclimation period).
- Weight at study initiation: 16.1-21.3 g (beginning of acclimation period).
- Housing: In groups of 4 (by dose), in cages with standards softwood bedding.
- Diet: Pelleted standard mouse maintenance diet, provided ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 6 days (18 September 2002 to 24 September 2002).

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

IN-LIFE DATES: From: 18 September 2002 To: 02 September 2002

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:water, 7:3 (v/v)
Concentration:
0.0 (vehicle control), 0.1, 1, 10 and 100%.
No. of animals per dose:
4
Details on study design:
MAIN STUDY
- Criteria used to consider a positive response: Firstly the criteria for a positive response is that one or more concentration of the test material should elicit a 3-fold or greater increase in the incorporation of ³H-methyl thymidine in comparison to the vehicle control, as indicated by the stimulation index. Secondly, that the data are compatible with a conventional dose response. Conversely, a test material which does not fulfil the above criteria is designated as unlikely to be a sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of test solution: The test material was placed in a volumetric flask and the vehicle (ethanol:water, 7:3 (v/v)) was quantitatively added. The weight volume dilutions were prepared individually using a magnetic stirrer as a homogenizer. Homogeneity of the test solution was maintained during treatment with the magnetic stirrer. Preparations were made shortly before each dosing. The test solutions were analysed at four selected consecutive concentrations.
- Administration: Test solutions were topically applied to the dorsum of each ear lobe (left and right) on three consecutive days at an application volume of 25 µL. The area was dried with hot air after application, to avoid loss of the test material.
- Post application procedure: Five days after the first topical application the mice were injected intravenously into a tail vein with 250 µL of radio-labelled thymadine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions (phosphate buffered saline) lymph node cells were prepared from pooled lymph nodes which were washed and incubated with 5% trichloroacetic acid overnight. The precipitates were then resuspended in 5% trichloroacetic acid and transferred to scintillation vials with 10 mL of scintillation cocktail. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintilation counter. The ß-scintilation counter expressed ³H-methyl thymidine incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated for bodyweights.

Results and discussion

Positive control results:
The stimulation indices of 2.9, 2.6 and 7.1 were determined with the positive control at concentrations of 5, 10 and 25% in acetone:olive oil, 4:1 (v/v). Based on the criteria, the test material was found to be a non-sensitiser when tested up to 10% (w/v) in acetone:olive oil, 4:1 (v/v). Alpha-hexylcinnamaldehyde showed an alergenic potency when tested at a concentration of 25% (w/v). EC3 is the estimated concentration for a stimulation index of 3. In this study an EC3 of 11.3% (w/v) was theoretically calculated with stimulation indices of 2.6 and 7.1 at positive control concentrations of 10% and 25% (w/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The application of the test material at concentrations of 0.1, 1, 10 % (w/v) in ethanol:water, 7:3 (v/v) and 100% (undiluted) resulted in a stimulation index which was less than 3-fold at all four concentrations, values can be seen in Table 1. Consequently, the test material was designated as unlikely to be a sensitiser under the conditions of the test.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The pooled disintegrations per minute and disintegrations per minute per lymph node are presented in Table 1 in the field "any other information on results incl. tables".

Any other information on results incl. tables

Viability/Mortality

- No deaths occurred during the study.

Clinical Signs

- No test material related clinical signs were observed in all the animals of the control group and those exposed to 0.1, 1 and 10% dilutions of the test material.

- Approximately 1 hour after the first topical application, a moderate swell was observed at both sites in all mice exposed to the test material at 100%. This lasted for four days and then reduced slightly, effects persisted until termination.

Bodyweights

- Bodyweights were within the range commonly recorded for animals of this strain and age.

Table 1: Results

Test item concentration % (w/v)   Meaurement dpm Calculation Result
dpm - BGa Number of lymph nodes dpm per lymph nodeb S.I.
BG I 3
BG II 3
CG 1 2345 2342 8 293
0.1 TG 2 2278 2275 8 284 1.0
1 TG 3 2746 2743 8 343 1.2
10 TG 4 2493 2490 8 311 1.1
100* TG 5 3720 3717 8 465 1.6
Positive control concentration % (w/v)            
BG I 3
BG II 3
CG 1 4232 4229 8 529
5 TG 2 121669 12166 8 1521 2.9
10 TG 3 10977 10974 8 1372 2.6
25 TG 4 29856 29853 8 3732 7.1

* undiluted as delivered by the sponsor.

BG = Background (1mL 5% trichloroacetic acide) in duplicated

CG = Control group

TG = Test group

S.I. = Stimulation Index

a = The mean value was taken from the figures BG I and BG II

b = Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was determined by dividing the measured value by the number of lymph nodes pooled.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test the stimulation index was less than 3-fold that of the control at all test concentrations and therefore, the test material is considered to be unlikely to be a skin sensitizer.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standard guidelines OECD 429 and OECD 406 using the mouse Local Lymph Node Assay (LLNA). The test material was applied as 0.1, 1, 10 % w/v preparation in ethanol:water, 7:3 (v/v) and 100% (undiluted). A vehicle control was run concurrently. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for the study.

Under the conditions of the test, the stimulation index induced by the test material was less than 3 -fold that of the control at all test concentrations, and therefore the test material is considered to be unlikely to be a skin sensitizer.