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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 17 May 2010 and 02 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
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)
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, 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 UK Limited, 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: Ethanol/distilled water 7:3. Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to the test material at concentrations of 10%, 5% or 2.5% w/w 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 10% w/w in ethanol/distilled water 7:3 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 10%, 5% or 2.5% 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 used at concentrations of 10%, 5% or 2.5% w/w in ethanol/distilled water 7:3. This vehicle was
chosen as it produced the most suitable formulation at the required concentration.

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:
None provided.
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 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.

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 ethanol/distilled water 7:3 Stimulation Index (SI) Result
15 9.61 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the undiluted test material and the test material at concentrations of 10%, 5% and 2.5% w/w in ethanol/distilled water 7:3. 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.

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

10

S-1

18

18

0

0

0

0

0

0

0

0

0

0=      No signs of systemic toxicity

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

6537.75

817.22

na

na

2.5

5605.01

700.63

0.86

Negative

5

9657.33

1207.17

1.48

Negative

10

10966.24

1370.78

1.68

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

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

2.5

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

5

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

10

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

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

19

19

0

1-2

17

17

0

1-3

19

22

3

1-4

22

25

3

2.5

2-1

21

21

0

2-2

21

20

-1

2-3

19

19

0

2-4

20

20

0

5

3-1

19

20

1

3-2

20

20

0

3-3

19

19

0

3-4

22

22

0

10

4-1

21

20

-1

4-2

19

19

0

4-3

18

19

1

4-4

19

19

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 Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26thmeeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

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

Project number :        0039/1138

Study dates :         12 February 2010 to 18 February 2010

Methods. 

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

9.61

Positive

Conclusion. 

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

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:

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 10w/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 asolutioninethanol/distilled water 7:3at concentrations of10%,5% or 2.5w/w. A further group of four animals was treated withethanol/distilled water 7:3alone.

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
ethanol/distilled water 7:3

Stimulation Index

Result

2.5

0.86

Negative

5

1.48

Negative

10

1.68

Negative

Conclusion. 

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

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

The substances in the category are considered to be similar on the basis that they have common structures of a lithium ion varying only by the length of the fatty acid chain and the presence of unsaturated and/or hydroxyl functional groups. As a result it is expected that the substances will have similar, predictable properties. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The fatty acid components of the category members are therefore not expected to be hazardous. As all category members are lithium salts, any toxicity is expected to be driven by the lithium ion. Due to the close structural similarity and the narrow range of carbon chain numbers covered in this category, the sensitisation potential is expected to be similar across the category.

Although fatty acids C18 (unsaturated) lithium salts is not in the list of substances being registered, this substance falls within the definition of the lithium salts of fatty acids C14-C22 category (see Appendix 1 – Category Justification Document) by virtue of its chemical structure and therefore read across from data on fatty acids C18 (unsaturated) lithium salts to other members of the category is considered to be justified (see below).

C18 (unsaturated) lithium salts was subjected to a Local Lymph Node Assay in mice according to OECD Guideline 429. The test material was considered to be a non-sensitiser under the conditions of the test with the highest Stimulation Index of 1.68 at 10% concentration in ethanol/distilled water.

A number of additional supporting studies on the skin sensitisation potential of lithium 12 -hydroxystearate in a grease base using the Magnusson and Kligman maximisation test have been considered. In all cases the results were negative. A published sensitisation study using a lithium complex grease containing 8.8% lithium 12 -hydroxystearate according to the Buehler method also showed no evidence of sensitisation.

On the basis of the category justification and the preamble to this endpoint, it is justifiable to read across the negative result from the fatty acids C18 (unsaturated) lithium salts study to the other members of the category, and together with the results from sensitisation studies on lithium 12-hydroxystearate support the lack of sensitisation potential of the lithium salts of fatty acids in the C14 – C22 category.


Migrated from Short description of key information:
Local Lymph Node Assay: Stimulation Index <3 at the highest concentration tested.

Justification for selection of skin sensitisation endpoint:
This substance is representative of the lithium salts of C14 - C22 fatty acids and can be read across to other category members

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification required. Stimulation Index <3 in a LLNA.