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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:
From 28 Jan 2004 to 07 Apr 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
(E)-3-methylcyclotetradec-5-en-1-one
Cas Number:
259854-70-1
Molecular formula:
C15H26O
IUPAC Name:
(E)-3-methylcyclotetradec-5-en-1-one
Constituent 2
Chemical structure
Reference substance name:
(Z)-3-methylcyclotetradec-5-en-1-one
Cas Number:
259854-71-2
Molecular formula:
C15H26O
IUPAC Name:
(Z)-3-methylcyclotetradec-5-en-1-one
Test material form:
liquid
Specific details on test material used for the study:
Description: Colourless to pale yellow liquid
Batch number: TQT0300497
Purity: 90.9%
Stability of test item: Stable under storage conditions
Expiry date: 28-NOV-2004
Storage conditions: In the original container at room temperature (20 °C ± 3 °C), away from direct sunlight.
Safety precautions: Routine hygienic procedures (gloves and goggles).

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognized as the recommended test system.
Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst/ The Netherlands
Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Body weight: 16 g - 24 g (ordered)
Identification: Each cage by unique cage card.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Room no. 129 B / RCC ltingen
Conditions: Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 ± 3 °C, relative
humidity 30 - 70 % and 10 - 15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
Accomodation: Individual in Makrolon type-2 cages with standard softwood bedding ("Ugnocel", Schill AG, CH-4132 Muttenz).
Diet:
Pelleted standard Kliba 3433, batch no. 78/03 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC)
Water: Community tap water from ltingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v)
No. of animals per dose:
4
Details on study design:
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 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µI, was spread over the entire dorsal surface (0 - 8 mm) 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 passed briefly over the ear's surface to prevent the loss of any of the test item applied.

Administration of 3H-Methylthymidine:
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µI of
79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tail vein.

Determination of incorporated 3HTDR:
Approximately five hours after treatment with 3HTdR all mice were euthanized with dry ice
(CO2).
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 two 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 for at least 18 hours 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 13-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The 13-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of raw data:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- 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.

Observations:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Twice daily from acclimatization start to the termination of in-life phase.
Body weights: Prior to the 1st application and prior to necropsy.
Clinical signs (local I systemic): Daily from acclimatization start to the termination of in-life phase. Particular attention was paid to the treatment sites.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 11.7 % (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Value:
16.4
Variability:
% (w/v)
Parameter:
SI
Value:
1.4
Test group / Remarks:
Group 2 - 1% in acetone:olive oil, 4:1 (v/v)
Parameter:
SI
Value:
1.8
Test group / Remarks:
Group 3 - 10% in acetone:olive oil, 4:1 (v/v)
Parameter:
SI
Value:
4.6
Test group / Remarks:
Group 4 - 25% in acetone:olive oil, 4:1 (v/v)
Cellular proliferation data / Observations:
Proliferation data: see table "Results from definitive test"
Viability/Mortality: No deaths occurred during the study period.
Clinical signs: No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group  3 (10 %). On the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for a total of three  days.
Body weights: The body weight of the animals, recorded prior to the1stapplication 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. Results from definitive test

Test item

concentration

% (w/v)

 

Measurement dpm

Calculation

Result

dpm -BGa>

number of lymph nodes

dpm perlymph nodebl

S.I.

--

BG I

10

--

--

--

--

--

BG11

3

--

--

--

--

--

CG 1

2201

2194

8

274

--

1

TG2

3097

3090

8

386

1.4

10

TG3

3937

3930

8

491

1.8

25

TG4

10007

10000

8

1250

4.6

BG=Background (1 ml 5 % trichloroacetic acid) in duplicate CG = Control Group

TG = Test Group

S.I.= Stimulation Index

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

b)      = Since the lymph nodes of the animals of a dose group were pooled,   DPM/nodewasdetermined by dividing the measured value by the number of lymph nodespooled

The proliferative capacity of pooled lymph node cells was determined by the incorporation  of3H-methyl thymidine measured on a 13-scintillation counter.

 

Test item concentration

%(w/v)

S.I.

Group 2

1

1.4

Group 3

10*

1.8*

Group 4

25*

4.6*

EC3=16.4 %(w/v)

A dose-response relation was observed.

*This value was used in calculation of EC3.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In this study STIMULATION INDICES of 1.4, 1.8 and 4.6 were determined with the test item at concentrations of 1 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
The test item KARMALONE was found to be a skin sensitizer and an EC3 value of 16.4 % (w/v) was derived.
Executive summary:

In order to study a possible contact allergenic potential of KARMALONE, three groups each of four female mice were treated daily with the test item at concentrations of 1 %, 10 % and 25%(w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil,4:1(v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine {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 of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in a ­ scintillationcounter.

No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group 3 (10 %). On the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for a total of threedays.

All treated animals survived the scheduled study period.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. The estimated concentration of test item required to produce a S.I.of 3 is referred to as the EC3 value.

 

Test item concentration

%(w/v)

S.I.

Group 2

1

1.4

Group 3

10*

1.8*

Group 4

25*

4.6*

EC3=16.4 % (w/v)

A dose-response relation was observed.

*This value was used in calculationofEC3.

In this study STIMULATION INDICES of 1.4, 1.8 and 4.6 were determined with the test item at concentrations of 1 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1(v/v).

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

The test item KARMALONE was found to be a skin sensitizer and an EC3 value of 16.4 % (w/v) was derived.