Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 13 to April 01, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 10, 2012/ signed on November 30, 2012)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Physical state: Clear colourless liquid
- Storage condition of test material: Room temperature 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 Limited, Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014 Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light


IN-LIFE DATES: From: March 13, 2014 To: April 01, 2014

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 25 and 50 % v/v in acetone/olive oil 4:1 and undiluted test item (100 % )
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Undiluted test item was used (100 %)
- Irritation: No signs of systemic toxicity, local irritation or irritation indicated by ≥25% increase in mean ear thickness noted.
Based on this information the undiluted test item and the test item at concentrations of 25 and 50 % v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Additional investigation: Measurement of ear thickness - The ear thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation) pre-dose and 1 hour post dose on Day 1, 1 hour post dose on Days 2 and 3 and on Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 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.
- Criteria used to consider a positive response: 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:
All formulations were used within two hours of preparation and assumed to be stable for this period. The concentration and homogeneity of the formulations were not determined by analysis.
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.
Probability values (p) were presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Results and discussion

Positive control results:
A group of five animals was treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25 % v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 8.42, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation index for 25, 50 and 100 % were 3.31, 5.31 and 10.12, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM / animal for vehicle, 25, 50 and 100 % were 2412.28, 7988.01, 12805.09 and 24413.20, respectively.

Any other information on results incl. tables

EC3 value

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 22 %.

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. 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.

 

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

See the attached document for information on tables of results

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test material is classified as a skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca (CBA/CaOlaHsd) strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity were noted with undiluted test item, 100 % was selected as the highest dose to be investigated in the main test.

Three groups, each of five animals, were treated with 50 μl (25 μl per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 100 %, 50 % or 25 % v/v for 3 consecutive days. A further group of five animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-Methyl Thymidine.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 to 6.

The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration

Mean dpm/animal

Stimulation Index

Result

Vehicle

2412.28

N/A

N/A

25 %

7988.01

3.31

Positive

50 %

12805.09

5.31

Positive

100 %

24413.20

10.12

Positive

 

The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 22 %.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 8.42, when tested at 25 % v/v. The test system was therefore considered to be valid.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated. Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Under the test conditions, test material is classified as a skin sensitizer in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.