Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 29 December 2009 and 14 January 2010.
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

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)
Remarks:
Date of inspection: 15/09/09 Date of signature: 26/11/09
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification : PDE-28
Description : red powder
Batch number : 1RF-8017
Date received : 23 March 2009
Expiry date : not available
Storage conditions : 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:
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):
Food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.

- Water (e.g. ad libitum):
Free access to mains tap water was allowed throughout the study.

- 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

Study design: in vivo (LLNA)

Vehicle:
other: ethanol/distilled water (7:3)
Concentration:
For the purpose of the study, the test material was freshly prepared as a solution in ethanol/distilled water 7:3.
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. Red coloured staining on the ears was noted on Days 2 to 5.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in ethanol/distilled water 7:3.

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 freshly prepared as a solution in ethanol/distilled water 7:3. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

The test material was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

The preliminary screening test showed no signs of systemic toxicity. Red coloured staining on the ears was noted on Days 2 to 5.
Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in ethanol/distilled water 7:3.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None provided.

Results and discussion

Positive control results:
Method:
A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in ethanol/distilled water 7:3 at a concentration of 15% v/v. A further control group of five animals was treated with ethanol/distilled water 7:3 alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in ethanol/distilled water 7:3 Stimulation Index Result
15 10.68 Positive

Conclusion: α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
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 ethanol/distilled water 7:3 Stimulation Index Result 10 1.04 Negative 25 0.88 Negative 50 1.28 Negative
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.

Any other information on results incl. tables

Clinical Observations and Mortality Data:

Individual clinical observations and mortality data for test and control animals are given in Table 3.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Red coloured staining on the ears was noted, post dose on Day 1 and on Days 2 to 6, in all test animals.

   Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1.

Estimation of the Proliferative Response of Lymph Node Cells:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table2.

Bodyweight:

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

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.

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in
ethanol/
distilled water 7:3

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

50

S-1

21

21

0

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0


0=      No signs of systemic toxicity

Fs =    Red coloured staining on the ears

Table 2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
ethanol/
distilled water 7:3

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

7963.63

995.45

na

na

10

8305.62

1038.20

1.04

Negative

25

6980.93

872.62

0.88

Negative

50

10178.66

1272.33

1.28

Negative


dpm=  Disintegrations per minute

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


Table 3              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) in
ethanol/
distilled water 7:3

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

10

2-1

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

2-2

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

2-3

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

2-4

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

25

3-1

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

3-2

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

3-3

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

3-4

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

50

4-1

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

4-2

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

4-3

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

4-4

0

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs

0Fs



0=      No signs of systemic toxicity

Fs =    Red coloured staining on the ears

Table 4              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
ethanol/
distilled water 7:3

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

18

19

1

1-2

19

20

1

1-3

19

19

0

1-4

18

18

0

10

2-1

21

20

-1

2-2

20

21

1

2-3

19

21

2

2-4

20

20

0

25

3-1

20

21

1

3-2

17

19

2

3-3

19

20

1

3-4

21

18

-3

50

4-1

19

20

1

4-2

19

20

1

4-3

20

20

0

4-4

19

18

-1



Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a non-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 of 100% v/v, 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 a solution in acetone/olive oil 4:1at concentrations of 50% or 25% v/v. A further group of four animals was treated with acetone/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

1.16

Negative

50

0.87

Negative

100

1.05

Negative

Conclusion. 

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