Registration Dossier

Administrative data

Description of key information

One key study (Bradshaw, 2010) is available for the skin sensitisation endpoint. This study is considered to be a Reliability 1 study as it has been conducted to the appropriate guideline (OECD 249, Local Lymph Node Assay) and under the conditions of GLP.

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 07 December 2009 and 22 December 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 GLP inspection: 01 April 2010 Date of Signature on GLP certificate: 26 November 2009
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other:
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:
other: 1% pluronic L92 in distilled water. Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 25%, 10% or 5% w/w (in 1% pluronic L92 in distilled water)
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 test material at a concentration of 25% w/w in 1% pluronic L92 in distilled water, 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%, 10% and 5% w/w in 1% pluronic L92 in distilled water.

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 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above.

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)
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 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water 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 10.91 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.
EXAMPLE
Key result
Parameter:
SI
Value:
1.18
Test group / Remarks:
5% w/w in vehicle
Key result
Parameter:
SI
Value:
0.92
Test group / Remarks:
10% w/w in vehicle
Key result
Parameter:
SI
Value:
0.85
Test group / Remarks:
25% w/w in vehicle

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

Concentration (%w/w) in
1% pluronic L92in distilled water

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

25

S-1

18

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
(%w/w) in
1% pluronic L92 in distilled water

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

8803.21

1100.40

na

na

5

10415.78

1301.97

1.18

Negative

10

8073.28

1009.16

0.92

Negative

25

7473.49

934.19

0.85

Negative

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
(% w/w) in
1% pluronic L92 in distilled water

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

5

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

10

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

25

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

Table4              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
1% pluronic L92 in distilled water

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

18

19

1

1-2

17

17

0

1-3

19

19

0

1-4

18

19

1

5

2-1

19

18

-1

2-2

22

20

-2

2-3

18

17

-1

2-4

20

19

-1

10

3-1

17

17

0

3-2

18

18

0

3-3

17

18

1

3-4

19

20

1

25

4-1

18

19

1

4-2

18

18

0

4-3

21

20

-1

4-4

18

18

0

Current Positive Control Study for the Local Lymph Node Assay

Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of CommissionRegulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non‑Commission members of the European Centre for the Validation of Alternative Method (ECVAM) Scientific Advisory Committee (ESAC) at its 26thmeeting held on 26 – 27 April 2007 at ECVAM,,.

Test Material:α‑Hexylcinnamaldehyde, tech., 85%

Project number:        0039/1108

Study dates:              01 October 2009 to 07 October 2009

Methods. A group of five animals was treated with 50 µl (25 µl per ear) ofα‑Hexylcinnamaldehyde, tech., 85%as a solution in1% pluronic L92 in distilled waterata concentration of 25% v/v. A further control group of five animals was treated with1% pluronic L92 in distilled wateralone. The control group was shared with Harlan Laboratories Project number 0545/0744.

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
1% pluronic L92 in distilled water

Stimulation Index

Result

25

5.11

Positive

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

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.

This study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement for this endpoint. In addition, this study is considered to be adequate for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
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 of25w/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 asolution/suspensionin1% pluronic L92 in distilled waterat concentrations of25%,10% or5w/w. A further group of four animals was treated with1% pluronic L92 in distilled wateralone.

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
1% pluronic L92 in distilled water

Stimulation Index

Result

5

1.18

Negative

10

0.92

Negative

25

0.85

Negative

Conclusion. The test material was considered to be anon-sensitiserunder the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The study reports that trisodium trimetaphosphate is a non-sensitiser under the conditions of the study and as such is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).