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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
In vivo done previously (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 05 May 2009 and 27 May 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2009

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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):
- Physical state: pale yellow viscous liquid (4°C)
- Lot/batch No.:

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid floor polypropylene cages furnished with softwood flakes.
- Diet (e.g. ad libitum): 2014 Teklad global rodent diet, ad libitum
- Water (e.g. ad libitum):mains tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark cycle


IN-LIFE DATES: No data available

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10%, 25%, 50%
No. of animals per dose:
4 mice per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the substance was considered suitable for dosing in acetone/olive oil (4:1)at a concentration of 50% as upon mixing by vortex mixer the formulation formed a solution which could be administered by micropipette.
- Irritation: None noted
- Lymph node proliferation response: Expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ration of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: “Skin Sensitisation: Local Lymph Node Assay” / “Skin Sensitisation (Local Lymph Node Assay of Commission directive 2004/73/EC”
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ^3HTdR incorporation compared to control values.


TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. (50%)
Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was interpreted using GraphPad PRISM version 3.03.

Results and discussion

Positive control results:
Concentration v/v in acetone/olive oil 4:1: 15%
Stimulation Index: 8.34
Result: Positive

In vivo (LLNA)

Results
Key result
Parameter:
EC3
Value:
9.39

Any other information on results incl. tables

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in

Acetone/olive oil 4:1

dpm

dpm/Nodea

Stimulation Indexb

 

Result

Vehicle

9376.99

1172.12

na

na

10

29437.64

3679.71

3.14

Positive

25

74447.31

9305.91

7.94

Positive

50

178410.2

22301.28

19.03

Positive

 

Dpm = disintegrations per minutes

a = total number of lymph nodes per animals is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not Applicable

 

Individual clinical Observations and Mortality Data

 

Conc (%v/v) in acetone/olive oil 4:1

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-dose

Post-dose

Pre-dose

Post-dose

Pre-dose

Post-dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

10

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

25

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

50

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

 

0 = No signs of systemic toxicity

 

 

Individual Bodyweights and Bodyweight Changes

 

Conc (%v/v) in acetone/olive oil 4:1

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

 

 

Vehicle

1-1

20

20

0

1-2

19

19

0

1-3

21

20

-1

1-4

20

21

0

 

 

10

2-1

20

20

0

2-2

20

20

0

2-3

22

23

1

2-4

20

19

-1

 

 

25

3-1

18

17

-1

3-2

18

18

0

3-3

22

21

-1

3-4

18

17

-1

 

 

50

4-1

19

18

-1

4-2

20

19

-1

4-3

18

17

-1

4-4

21

20

-1

 

 

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Migrated information
Conclusions:
In a Local Lymph Node Assay in the Mouse, the substance initiated a Stimulation Index of greater than 3 at concentrations of 10, 25 and 50% and thus is considered to be a skin sensitizer under the conditions of the test.
EC3 value was determined to be 9.39%. Per the CLP guidance, substances are to be classified as skin sensitization 1A when the EC3 value is less than 2% and are to be classified as skin sensitization 1B when the EC3 value is greater than 2%.
Executive summary:

Introduction.A study was performed to assess the skin sensitization potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements OECD 429 and Method B42.

Methods.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 material 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.

Results.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:

Concentration (% w/w) in

acetone/olive oil 4:1

Stimulation Index

Result

 

10

3.14

Positive

25

7.94

Positive

50

19.03

Positive

 

According to the calculation the concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was found to be 9.39%.

Conclusion.The test material was considered to be a sensitiser under the conditions of the test.

EC3 value was determined to be 9.39%. Per the CLP guidance, substances are to be classified as skin sensitization 1A when the EC3 value is less than 2% and are to be classified as skin sensitization 1B when the EC3 value is greater than 2%.