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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isoprenol
- Analytical purity: 98.4 area-%; dose calculation not adjusted to purity
- Lot/batch No.: 20614288Q0
- Expiration date of the lot/batch: January 31, 2011
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus, AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Mean weight (± SD) at study initiation: 20.2 ± 1.4 g
- Housing: Single caging
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories B.V., Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum,
- Acclimation period: At least 5 days prior to the start of dosing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 20-65%
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, and 100% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone:olive oil (4+1). Vortexing was necessary to formulate the test item.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50% and 100% each on three consecutive days. At the tested concentrations the animals did not show any signs of local irritation as confirmed by the ear thickness and ear weight measurements. Also, animals did not show any signs of systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Administration of ³H-Methyl Thymidine
Five days after the first topical application, all mice were administered with 250 µl of 79.4 µCi/ml ³HTdR (corresponds to 19.9 µCi ³HTdR per mouse) by intravenous injection via a tail vein.
- Determination of lymph node weights
For the determination of lymph node weight, after excision, the lymph nodes were pooled per animal and weighed immediately after removal using an analytical balance.
- Determination of incorporated ³HTdR
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After further processing, the level of ³HTdR incorporation was measured on a ß-scintillation counter as the number of radioactive disintegrations per minute (DPM).
- Determination of Ear Weights
After the lymph nodes have been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal.

- 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 lymph nodes relative to that recorded for control lymph nodes (stimulation index). 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 sensitiser 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 substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
The ANOVA (Dunnett-test) was conducted on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Positive control substance: alpha-Hexylcinnamaldehyde
Test item concentration % (w/v) S.I.
0 1.00
5 1.21
10 2.09
25 6.22

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: SI
Remarks on result:
other: see Remark
Remarks:
Test item concentration % (w/v) SI 0 - 25 1.48 50 1.67 100 2.28 None of the tested concentrations induced an S.I. greater than 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test item concentration % (w/v) Measurement DPM 0 4785 25 7070 50 7966 100 10898
Key result
Parameter:
SI
Value:
1.48
Test group / Remarks:
25 % test group
Key result
Parameter:
SI
Value:
1.67
Test group / Remarks:
50 % test group
Key result
Parameter:
SI
Value:
2.28
Test group / Remarks:
100 % test group

Any other information on results incl. tables

Further results:

- No deaths occurred during the study period.

- No symptoms of local irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

- The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

- A relevant increase in ear weights was not observed.

- A statistically significant increase in lymph node weights was observed in the groups treated with 50 and 100% test item concentration in comparison to the vehicle control group (p=0.027):

Table of Lymph Node Weights

Animal No.

Concentration
% (w/v)

Lymph Node Weights after Necropsy

 

Lymph Node Weight (mg per animal)

Mean Lymph Node Weight (mg per animal)

SD

1

0

3.90

4.45

0.61

2

4.30

3

4.27

4

5.32

5

25

5.04

5.43

0.65

6

5.80

7

4.73

8

6.13

9

50

5.91

6.11*

0.73

10

5.27

11

7.02

12

6.25

13

100

4.73

6.28*

1.10

14

6.71

15

6.36

16

7.30

* statistically significant increase vs. control group (p=0.027)

SD= Standard Deviation

Applicant's summary and conclusion