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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 02 November 2009 and 24 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
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)
Remarks:
Date of GLP inspection: 12 January 2010 Date of Signature on GLP certificate: 26 November 2009
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited, Bicester, Oxon, UK.

- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23g.

- Housing:
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet (e.g. ad libitum):
ad libitum (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK)

- Water (e.g. ad libitum):
ad libitum.

- Acclimation period:
At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
The temperature was controlled to remain within the target ranges of 19 to 25 deg C.

- Humidity (%):
The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr):
The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES:
From: Day 1 To: Day 6
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
EXAMPLE: Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 100% (undiluted), 50% or 25% v/v (in acetone/olive oil 4:1)
No. of animals per dose:
Groups of four mice were treated
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are give in the results section (table 1).

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 25% and 50% v/v in acetone/olive oil 4:1 and 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- 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 lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

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. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was used undiluted and also freshly prepared in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above.

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 Guidelines.

The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (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, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
N/A
Positive control results:
One group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

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

Concentration % v/v in acetone/olive oil 4:1 Stimulation Index (SI) Result
15 3.12 Positive


Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A stimulation index of less than 3 was recorded for the test material at a concentration of 25% v/v in acetone/olive oil 4:1. A stimulation index of greater than 3 was recorded for the undiluted test material and the test material at a concentration of 50% v/v in acetone/olive oil 4:1. The stimulation index (SI) results are given in Table 2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Table1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

100%

S-1

19

19

0

0

0

0

0

0

0

0

0

0=      No signs of systemic toxicity

Table2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

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

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

10453.79

1306.72

na

na

25

17547.42

2193.43

1.68

Negative

50

36209.14

4526.14

3.46

Positive

100

33894.66

4236.83

3.24

Positive


dpm=  Disintegrations per minut

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

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

na =    Not applicable

Table3              Individual Clinical Observations and Mortality Data

Concentration
(% 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

25

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

50

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

100

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


0=      No signs of systemic toxicity

Table 4              Individual Bodyweights and Bodyweight Changes

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

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

22

21

-1

1-2

18

18

0

1-3

21

20

-1

1-4

23

23

0

25

2-1

18

19

1

2-2

19

20

1

2-3

19

19

0

2-4

19

19

0

50

3-1

19

20

1

3-2

20

19

-1

3-3

18

18

0

3-4

18

19

1

100

4-1

18

18

0

4-2

16

18

2

4-3

18

18

0

4-4

18

19

1

Table 5              Positive Control

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

Stimulation Index

Result

15

3.12

Positive

EC3 VALUE

EC3= c + [[(3-d)/(b-d)] x (a-c)]

a =     50
b =     3.46
c =     25
d =     1.68

EC3=25+ [[(3-1.68)/(3.46-1.68)] x (50-25)] =43.5


a= lowest concentration giving stimulation index >3

b = actual stimulation index caused by a

c = highest concentration failing to produce a stimulation index of 3

d = actual stimulation index caused by c

The concentration of test material expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 43.5%v/v in acetone/olive oil 4:1.

Interpretation of results:
sensitising
Remarks:
Migrated information The concentration of test material expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 43.5%v/v in acetone/olive oil 4:1.
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test.
Executive summary:

Introduction. A study was performed to assess the skin sensitisation 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 of the following:

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of100%, 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 undiluted test material or the test material as asolutioninacetone/olive oil 4:1at concentrations of50% or25v/v. A further group of four animals was treated withacetone/olive oil 4:1alone.

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

Stimulation Index

Result

25

1.68

Negative

50

3.46

Positive

100

3.24

Positive

The concentration of test material expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 43.5%v/v in acetone/olive oil 4:1.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test substance was tested for skin sensitization potential using a mouse Local Lymph Node Assay (LLNA). There was evidence of induction of a lymphocyte proliferative response, indicative of skin sensitization. The stimulation index (SI) was reported to exceed 3 at concentrations of 50 and 100% test material and the EC3 value was calculated to by 43.5%. Accordingly, it is classified and labelled as may cause an allergic skin reaction (CLP Category 1).


Migrated from Short description of key information:
A positive response was observed in the Local Lymph Node Assay for the test substance.

Justification for selection of skin sensitisation endpoint:
Only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test data support classification of the registered substance as a skin sensitiser (Category 1) according to the criteria laid down in Regulation (EC) No 1272/2008 (i.e. CLP)

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