Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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)
Remarks:
(RCC - Cytotest Cell Research GmbH)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Biofert Plusz
Details are presented in "Confidential details on test material"

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 19.6 to 23.3 g
- Housing: individual in Macrolon Type I
- Diet: certified standard pellets ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days prior to first dosing


ENVIRONMENTAL CONDITIONS
- according to guideline


IN-LIFE DATES:
From April 08, 2008, to April 23, 2008

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
The test item in the main study was assayed at 0, 25, 50 and 100% of the original liquid substance (0, 7.03, 14.05 or 28.1% dry mass)
No. of animals per dose:
4 females in the main study
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: After a solubility experiment, the highest concentration which could be technically used was a 50% (14.05% dry mass) suspension in propylene glycol. Upon sponsors consent propylene glycol was used as a vehicle.
- Irritation: A pre-test was performed in two mice with concentrations of 10, 25, 50 and 100% of the original liquid substance (2.81, 7.03, 14.05, or 28.1% dry mass, respectively) on one ear each on three consecutive days (clinical signs scored 24 h after each application). After the second application and onwards the highest dose (100%) induced redness of the ear skin. All other concentrations did not induce any signs of irritation or systemic toxicity



MAIN STUDY
- Criteria used to consider a positive response:
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 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 for either local or toxicological response.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice (n=4 per group) was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentartions. The application volume (25 µL) was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. Control animals were treated with the vehicle only.
Five days after the first topical application, all mice were administered with 250 µL of 81.6 µCi/mL 3HTdR (3H-methyl thymidine) by intravenous injection via tail vein. Approximately five hours after treatment with 3HTdR all mice were necropsied and the draining lymph nodes were excised and pooled per group (8 nodes per group). The level of 3HTdR incorporation was than measured on a beta-scintillation counter. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated in body weight tables

Results and discussion

Positive control results:
A GLP study with a positive control substance (alpha-Hexylcinnamaldehyde) was performed with test item concentrations (w/v) of 5, 10 and 25% in an acetone:olive (4:1) vehicle in February 2008 and included as reference into this study. The outcome of the study demonstrated the sensitivity and validity of the test system.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation Index (S.I.) was 1.99, 2.18 and 1.47 for the three Biofert Plusz dose groups.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM per lymph nodes was 339.0, 674.4, 738.1 and 497.5 for the control group and the three dose groups, respectively.

Any other information on results incl. tables

No clinical signs were observed in any dose group. The body weight was within the common range.

 Table 1: Calculation and Summary of Individual Data

 

Test item concentration

% (w/v)

Group

Measurement

DPM

Calculation

Result

DPM-BGa)

Number of lymph nodes

DPM per lymph nodeb)

S.I.

--

BG I

30

--

--

--

--

--

BG II

22

--

--

--

--

--

1

2738

2712

8

339.0

--

25

2

5421

5395

8

674.4

1.99

50

3

5931

5905

8

738.1

2.18

100

4

4006

3980

8

497.5

1.47

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

1 = Control group

 2-4 = test groups

S.I = Stimulation Index

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

b) = Since the lymph nodes 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
Conclusions:
None of the tested concentrations induced an S.I. greater than 3. Therefore, an EC3 value could not be calculated. Thus the test item Biofert Plusz was found not to be a skin sensitiser under the described conditions.