Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/JIbm from RCC Ltd, Laboratory Animal Service, Füllinsdorf, Switzerland
- Age at study initiation: 8-12 weeks (beginning of acclimatisation)
- Weight at study initiation: 16-24g (ordered)
- Identification: Each cage by unique cage card, in every cage each animal by individual code marked at tail with a permanent pen.
- Randomisation: Randomly selected by computer algorithm at time of delivery.
- Housing: In groups of four in Makrolon type-3 cages with standard softwood bedding.
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no 54/03 mouse
maintenance diet available ad libitum. Results of analyses for contaminants are archived at the laboratoy.
- Water (e.g. ad libitum): Community tap water, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at the laboratory.
- Acclimation: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Air-conditioned with target ranges for room temperature 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:
unchanged (no vehicle)
Concentration:
100 % (w/v) (undiluted as delivered by the sponsor)
No. of animals per dose:
4 females
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
TREATMENT PREPARATION AND ADMINISTRATION:
TOPICAL APPLICATION
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear lobe (left and right) with the undiluted test item. The application volume, 25 µL, was spread over the entire dorsal surface (ø~8 mm) of each ear lobe once daily for three consecutive days. A negative control group of mice remained untreated. The vehicle and positive control groups of mice were separately treated with an equivalent volume of the relevant vehicle alone or with a same volume of the positive control item dilution. A hair dryer was passed briefly over the ear‘s surface to prevent the Ioss of any of the test items applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE
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 250 µL of 76.6 µCi/ml 3HTdR (equal to 19.1 µCi 3HTdR) by intravenous injection via a tail vein.
DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanised 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 twice 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 β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-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.

POSITIVE CONTROL:
The contact allergenic potential of ALPHA-HEXYLCINNAMALDEHYDE was assessed in three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % 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 of 3H-methyl thymidine measured in a β-scintillation counter.
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.

Results and discussion

Positive control results:
No test item-related clinical signs were observed in any animals of the control group, Group 2 (5%) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %)‚ persisting for four days. On the third application day, a slight ear swelling was also observed at both dosing sites in all mice of Group 4 (25 %)‚ persisting for the remainder of the in-life phase of the study.
All treated animals survived the scheduled study period.
S.I. of 1.5, 3.2 and 6.9 were determined with the test item at oncentrations of 5%, 10% and 25% (w/v) in acetone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitiser 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 S.I.; the test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitiser and an EC3 value of 9.4 % (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
3
Test group / Remarks:
test item group (100 %)
Parameter:
SI
Value:
3.5
Test group / Remarks:
positive control group (25%)
Parameter:
other: disintegrations per minute (DPM)
Value:
9 051
Test group / Remarks:
test item group (100 %)
Remarks on result:
other: each based on 8 lymph nodes investigated; dpm per lymph node 1131
Parameter:
other: disintegrations per minute (DPM)
Value:
11 962
Test group / Remarks:
positive control group (25%)
Remarks on result:
other: each based on 8 lymph nodes investigated; dpm per lymph node 1495

Any other information on results incl. tables

1. Calculation and Results of Individual Data

The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a β-scintillation counter. The following resuits were obtained:

Vehicle HCA: acetone:olive oil, 4:1 (v/v)

 Substance     % (w/v)          Measurement   DPM  Calculation        Result
 DPM - BG a)  number oflymph nodes  DPM perlymph node b)  S.I.
 --    BG I  0  --  --  --  --
 --  --  BG II  0  --  --  --  --
 --  --  CG1  ***  ***  ***  377***  --
 --  --  CG2  3398  3398  8  425  --
 HCA (PC)  25  TG3  11962  11962  8  1495  3.5 c)
 Test Item  100*  TG11  9051  9051  8  1131  3.0 d)

* Undiluted as delivered by the Sponsor

*** The value is basis on the historical data:

(M±SD = 377±79; Group Number = 9; Mice Number = 36).

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 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

c) = CG2 is used as the control group to calculate result

d) = CG1 is used as the control group to calculate results

2. Viability / Mortality

No deaths occurred during the study period.

3. Clinical Signs

No test item-related clinical signs were observed in any animals of the two control groups (Groups 1 and 2).

Approximately one hour after the first topical application, a slight to moderated ear erythema was observed at both dosing sites in all mice of the positive control Group 3 (HCA, 25 %)‚ persisting for a total of four days. In addition, on the second application day, a slight ear swelling was observed at both dosing sites in all mice of this group, persisting for a total of three days.

On the third or fourth application days, a slight to severe head and both ears hair loss was observed in all mice of test Group 11 (test item), persisting for the remainder of the in-life phase of the study.

4. Body Weights

The body weight of the animals, recorded prior to the 1st application 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:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test substance was found to be a skin sensitisers under the conditions of the test.
Executive summary:

In order to study a possible contact allergenic potential of the test item (and several other substances), eight groups each of four female mice were treated daily with the test items by topical application to the dorsum of each ear lobe (left and right) for three consecutive days in a GLP study according to OECD Guideline 429. A negative control group of four mice remained untreated. A positive control group of four mice was treated with Alpha-hexylcinnamaldehyde (HCA) at concentration of 25% (w/v) in acetone:olive oil, 4:1 (v/v). Five days after the first topical application the mice were injected intravenously into a tail vain 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 of 3H-methyl thymidine measured in a β-scintillation counter.

No test item-related clinical signs were observed in any animals of the two control groups (Groups 1 and 2).

Approximately one hour after the first topical application, a slight to moderated ear erythema was observed at both dosing sites in all mice of the positive control Group 3 (HCA, 25 %)‚ persisting for a total of four days. In addition, on the second application day, a slight ear swelling was observed at both dosing sites in all mice of this group, persisting for a total of three days.

On the third or fourth application days, a slight to severe head and both ears hair loss was observed in all mice of test Group 11 (test item), persisting for the remainder of the in-life phase of the study. All treated animals survived the scheduled study period.

In this study S.I. of 3.5 and 3.0 were determined with the positive control item HCA at 25 % in acetone:olive oil, 4:1 (v/v) and the test item, respectively. A test item is regarded as a sensitiser in the LLNA, if the exposure to one er more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the S.I..

The positive control item HCA and the test item were found to be skin sensitisers.