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Diss Factsheets

Administrative data

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 14 September 2009 and 30 September 2009
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
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)
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:
Animals and Animal Husbandry
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Bicester, Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 10% , 5% or 2.5% v/v (in acetone/olive oil 4:1)
No. of animals per dose:
Groups of four mice were treated
Details on study design:
Procedure
Preliminary Screening Test
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 10% w/w in acetone/olive oil 4:1, 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.

Main Test
Test Material Administration
Groups of four mice were treated with the test material at concentrations of 10%, 5% or 2.5% w/w in acetone/olive oil 4:1. 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, 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.
A further group of four mice received the vehicle alone in the same manner.

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, 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.
Positive control results:
Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. This control group was shared with Project
number 0673/0012.

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 is as follows:

Concentration (% v/v) in
acetone/olive oil 4:1 Stimulation Index Result
15 3.70 Positive

Conclusion. α Hexylcinnamaldehyde 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 test material at concentrations of 10%, 5% and 2.5% v/v in acetone/olive oil 4:1. The stimulation index (SI) results are given in Table 2 - see section any other information on results.
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- see section any other information on results.
Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

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

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

21

21

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
acetone/olive oil 4:1

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

12222.07

1527.76

na

na

2.5

13722.06

1715.26

1.12

Negative

5

12251.10

1531.39

1.00

Negative

10

10496.22

1312.03

0.86

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
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

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
acetone/olive oil 4:1

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

19

18

-1

1-2

18

19

1

1-3

18

18

0

1-4

19

19

0

2.5

2-1

20

21

1

2-2

18

18

0

2-3

19

19

0

2-4

17

18

1

5

3-1

19

19

0

3-2

19

19

0

3-3

18

19

1

3-4

22

21

-1

10

4-1

18

18

0

4-2

21

21

0

4-3

20

19

-1

4-4

18

17

-1

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 24 April 2002)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 10% 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 asuspension/emulsioninacetone/olive oil 4:1at concentrations of 10%, 5% or 2.5% w/w. 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 (%w/w) in
acetone/olive oil 4:1

Stimulation Index

Result

2.5

1.12

Negative

5

1.00

Negative

10

0.86

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:

A skin sensitisation test to OECD test guideline 429 and to acceptable quality standards show that the registered substance in its isolated form is non-sensitising under the conditions of the test.


Justification for selection of skin sensitisation endpoint:
GLP compliant guideline study with a klimisch score of 1

Justification for classification or non-classification