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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
Study period:
27 July - 18 Aug 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

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
Type:
Constituent
Test material form:
other: solid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V.; AD Horst, The Netherlands
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 17.7 - 21.0 g
- Housing: in groups in Macrolon Type II / III cages with wire mesh top with granulated soft wood bedding
- Diet: pelleted standard diet (Harlan Laboratories B.V., Horst, The Netherlands), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/- 2°C
- Humidity (%): 45-77 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27.07. To: 18.08.2011

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5 or 10%
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 a 10 % solution in actone:olive oil (4+1).
At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicle nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C)
- Irritation: performed on two female mice, by topical application of concentrations of 5, and 10 % on each ear each on three consecutive days. Prior to application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch and were immediately pooled per animal and weighed using an analytical balance.
On day 3 and 4, the animal treatedwith 5 and 10% test item concentration showed reddening of the ear skin (score 2). On day 5, the animal treated with 10% test item concentration showed reddening of the ear skin (score 1). Thus, the test item in the main study was assayed at 2.5, 5 and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The sensitivity and reliability of the experimental technique employed was assessed by use of a
substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice (α-Hexylcinnamaldehyde in acetone:olive oil, 4:1 )

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl was spread to the entire dorsal surface of each ear of each mouse once daily for three consecutive days. On day 6 an injection of 250 µl phosphate buffered saline (PBS) containing20.1 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours
later, the draining Auricular lymph node of each ear was excised and pooled per group (8 nodes per group) into PBS. A single cell suspension of lymph node cells was prepared from each group. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials. The level of 3HTdR incorporation was then measured on a ß-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of Hexylcinnaldehyde which is known to have skin sensitisation properties in CBA/CaOlaHsd mice.
The EC3 value was calculated to be 12.1%.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.86
Test group / Remarks:
2.5 % in acetone:olive oil (4+1 v/v)
Key result
Parameter:
SI
Value:
2.21
Test group / Remarks:
5 % in acetone:olive oil (4+1 v/v)
Key result
Parameter:
SI
Value:
2.53
Test group / Remarks:
10 % in acetone:olive oil (4+1 v/v)

Any other information on results incl. tables

Table 1: Calculation and Results

Vehicle: acetone:olive oil (4+1 v/v)

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

27

---

---

---

---

---

BG II

22

---

---

---

---

0

1

1502

1478

7

211.1

1.00

2.5

2

3162

3138

8

392.2

1.86

5

3

3753

3729

8

466.1

2.21

10

4

4291

4267

8

533.3

2.53

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

1    =  Control Group

2-4=  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

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test itemHostanox SE 10 was found to be not a skin sensitiser under the described conditions.
Executive summary:

The sensitization potential was investigated according to OECD Guideline 429.

The test item concentrations chosen for the LLNA test were 2.5, 5, 10 %.

No deaths occurred during the study period. No systemic or local findings were observed during the study period. The body weight of the animals was within the range commonly recorded for animals of this strain and age.