Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-21 until 2014-06-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (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
Test material form:
solid: crystalline
Details on test material:
- Physical state: Yellowish crystals
- Purity test date: 10 October 2013
- Expiration date of the lot/batch: End September 2015
- Stability under test conditions: Stable until end September 2015
- Storage condition of test material: Room temperature (<30°C) in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Individually in suspended solid floor polypropylene cages with softwood woodflakes
- Diet: 2014C Teklad Global Rodent diet ad libitum. Harlan Laboratories UK Ltd., Oxon, UK
- Water: Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 May 2014 To: 11 June 2014

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50, 25, 10%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Test item was soluble in the vehicle.
- Method: One mouse was treated by daily application of 25 μL of the test item at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days. The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured pre-dose on Day 1, post dose on Day 3 and on Day 6. Mean ear thickness changes were calculated between Days 1-3 and 1-6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
- Irritation: No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION: A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear on days 1, 2 and 3. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% w/w. The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

OBSERVATIONS: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. A single cell suspension of the lymph node cells for each individual animal was prepared and 3HTdR incorporation was determined.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:
Not applicable

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 1.52, 1.60 and 2.15 for 10, 25 and 50% test item in acetone/olive oil 4:1, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 6530.36±1421.23, 9958.07±2038.31, 10461.65±2931.47, 14047.58±4146.80 for 0, 10, 25 and 50% test item in acetone/olive oil 4:1, respectively

Any other information on results incl. tables

Table 1: Individual disintegrations per minute (dpm) and Stimulation Indices

Concentration (%)

dpm/animal

mean dpm /animal ± SD

Stimulation index

Result

Vehicle

6954.18

6530.36±1421.23

N/A

N/A

8091.87

4680.09

6395.28

10

8234.02

9958.07±2038.31

1.52

Negative

11928.96

11504.53

8164.78

25

6928.82

10461.65±2931.47

1.60

Negative

13610.45

9367.00

11940.34

50

8529.24

14047.58**±4146.80

2.15

Negative

18121.30

16092.22

13447.54

** Significantly different from control group p<0.01

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, under the conditions of the present assay, the test item tested in a suitable vehicle was shown not to have skin sensitisation potential in the Local Lymph Node Assay.
Executive summary:

A study was performed to assess the skin sensitization potential of the test substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

No mortality or signs of systemic toxicity was observed for the test item treated animals during the study. No treatment related effects were observed on body weight. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 1.52, 1.60 and 2.15 for 10, 25 and 50% test item in acetone/olive oil 4:1, respectively.

In conclusion, under the conditions of the present assay, the test item tested in a suitable vehicle was shown not to have skin sensitisation potential in the Local Lymph Node Assay.