Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 31 May 2011 to 16 November 2011
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

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:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
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:
Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione
EC Number:
915-316-2
Molecular formula:
C50H80O4
IUPAC Name:
Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 8 weeks
- Weight (mean) at study initiation: 21.2 g +/- 0.8 g
- Fasting period before study: No
- Housing: The animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Techniplast 1145T, 435 cm2, 36.9 x 15.6 x 13.2 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained two enrichments (mouse hut and cocoon). In the main test, on day 6 before the 3H-TdR injections, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
- Diet (ad libitum): SSNIFF R/M-H pelleted diet, batch No. 9945009 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: 13 to 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES (Main test): From: 01 June 2011 to 21 June 2011

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0.5, 1, 2.5, 5 and 10% (w/v) in propylene glycol (non-homogeneous preparations were obtained at higher concentrations)
No. of animals per dose:
4 females/dose
Details on study design:
RANGE FINDING TESTS:
- Irritation:
A preliminary test was performed in four animals to assess the irritant potential of the test item (through ear thickness measurement). At the concentrations of 1, 2.5, 5 and 10% (highest technically applicable concentration), the test item did not show any signs of irritation. Therefore, in the main test, concentrations applied were 0.5, 1, 2.5, 5 and 10%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI). The test item is considered as a skin sensitizer when the SI for a dose group is > or = 3 together with consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the vehicle control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
Auricular Lymph Node Cell (ALNC) proliferation assay:
Lymph node cell proliferative response was measured as described by Kimber and Dearman (1). On day 6 of the main test, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
Approximately 5 hours later, the main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of ALNC was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.
Cell suspensions were washed once with 15 mL of 0.9% NaCl. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic (TCA) at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three milliliters of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using  scintillation counting.
The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. Stimulation Indices (SI) were calculated according to the following formula: SI = dpm per node of the treated group / dpm per node of the vehicle control group.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
According to the OECD guideline 429, a statistical analysis of the data is not mandatory.

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 7.38).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see summary table below.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see summary table below.

Any other information on results incl. tables

 Summary results of the proliferation assay:

Treatment and concentrations

Number of nodes per group

dpm per group

dpm per node

Stimulation index (SI)

Increase in ear thickness (% between day 1 and day 6)

Irritation level

ECs value

Vehicle

8

1011

126.38

/

-3.03

/

NA

Test item 0.5%

8

662

82.75

0.65

0.00

I

Test item 1%

8

799

99.88

0.79

-2.06

I

Test item 2.5%

8

833

104.13

0.82

5.10

I

Test item 5%

8

1019

127.38

1.01

-2.02

I

Test item 10%

8

278

34.75

0.27

1.01

I

HCA 25%

8

7461

932.63

7.38

/

/

 

NA = not applicable

dpm = disintegrations per minute

I = non-irritant (increase in ear thickness < 10%)

ECs value = theoretical concentration resulting in a SI value of 3

HCA = alpha-hexylcinnamaldehyde

Other observations:

No unscheduled deaths occurred during the main test. 

No clinical signs and no local reactions were observed in any animals

The body weight change of test item-treated animals was similar to that of control animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study the stimulation indices (S.I.) of 0.65, 0.79, 0.82, 1.01 and 0.27 were determined with the test item at concentrations of 0.5, 1, 2.5, 5 and 10% in propylene glycol. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than 3. The test material Rhodiastab 55P was found not to be a skin sensitiser under the described conditions.

The reaction mass of Stearoylbenzoylmethane and Palmitoylbenzoylmethane is not classified as a skin sensitiser according to the criteria of Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).
Executive summary:

In order to study a possible contact allergenic potential of Rhodiastab 55P, a local lymph node assay (LLNA) was performed according to the OECD Guideline 429 and under GLP regulations.

For this purpose, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 0.5, 1, 2.5, 5 or 10% (maximum technically applicable concentration) under a dose‑volume of 25 µL. One negative control group of four females received the vehicle (Propylene Glycol) under the same experimental conditions. Additionally, one positive control group of 4 females received the positive control,α‑hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.

From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded on days 1 and 6. After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI). 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.).

 

No unscheduled deaths occurred and no clinical signs were observed in any animals throughout the study. No relevant increase in ear thickness and no local reactions were reported.

In this study the stimulation index of 0.65, 0.79, 0.82,1.01and 0.27 were determined with the test item at concentrations of 0.5, 1, 2.5, 5 and 10% (w/v) in propylene glycol, respectively. The EC3 value could not be calculated since none of the tested concentrations induced a S.I. greater than 3. Rhodiastab 55P was therefore found to be a non-sensitizer when tested at up to the concentration of 10 %.

 

The reaction mass of Stearoylbenzoylmethane and Palmitoylbenzoylmethane is not classified as a skin sensitiser according to the criteria of Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).