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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:
15 April 2013 and 09 May 2013
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:
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:
Animals and Animal Husbandry

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages.The animals were nulliparous and non pregnant. After an acclimatization 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 (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and bedding were considered not to contain contaminants that could reasonably be expected to affect the purpose or integrity of 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)
Concentration:
Preliminary screening test
50% or 25% v/v in acetone/olive oil 4:1
Main test
25%, 10% and 5% v/v in acetone/olive oil 4:1
No. of animals per dose:
5
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6.

The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.


Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 25%, 10% or 5% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at a concentration of 25% v/v in acetone/olive oil 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.


3H-Methyl Thymidine Administration
Five days following the first topical application of the test item 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.


Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Ear Thickness Measurements: The ear thickness of each mouse was recorded on Day 1 (prior to dosing), Day 3 and Day 6 (prior to termination).

Ear Weight Measurements: The ear weight (ear punch) measurements of each mouse were performed on Day 6 after termination and excision of nodes.


Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4 C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by  scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical Analysis
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)
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 positive control 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
25 4.01 Positive

α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The individual DPM's are given in Table 4.
Key result
Parameter:
SI
Value:
ca. 4.01
Test group / Remarks:
alpha-Hexylcinnamaldehyde (25% v/v in acetone/olive oil 4:1) / positive control
Remarks on result:
other: positive
Key result
Parameter:
SI
Value:
ca. 0.99
Test group / Remarks:
Farnesene 5% v/v in acetone/olive oil 4:1
Remarks on result:
other: negative
Key result
Parameter:
SI
Value:
ca. 1.42
Test group / Remarks:
Farnesene 10% v/v in acetone/olive oil 4:1
Remarks on result:
other: negative
Key result
Parameter:
SI
Value:
>= 1.51
Test group / Remarks:
Farnesene 25% v/v in acetone/olive oil 4:1
Remarks on result:
other: negative

           

Individual clinical observations and mortality data for test and control animals are given in Table 5. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test

       

Individual body weights and body weight change for test and control animals are given in Table 6.

 

Body weight change of the test animals between Day 1 and Day 6 was comparable to those observed in the corresponding control group animals over the same period.

             

Table 4     Individual Disintegrations per Minute and Stimulation Indices

Concentration

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle

Acetone/olive oil 4:1

1-1

2661.62

2722.10
(±295.65)

na

na

1-2

2634.42

1-3

2980.13

1-4

3031.64

1-5

2302.69

Test Item

5% v/v in acetone/olive oil 4:1

2-1

2572.73

2707.34
(±716.18)

0.99

negative

2-2

3776.39

2-3

2353.40

2-4

2963.38

2-5

1870.78

Test Item

10% v/v in acetone/olive oil 4:1

3-1

747.09

3861.38
(±2221.23)

1.42

negative

3-2

2964.58

3-3

3809.57

3-4

5194.22

3-5

6591.45

Test Item

25% v/v in acetone/olive oil 4:1

4-1

4339.77

4106.38
(±644.98)

1.51

negative

4-2

3024.35

4-3

4750.83

4-4

4244.36

4-5

4172.58

Positive Control Item

25% v/v in acetone/olive oil 4:1

5-1

9342.27

10903.77*
(±2796.80)

4.01

positive

5-2

12545.31

5-3

9551.35

5-4

14969.02

5-5

8110.91

 

The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups.


dpm=      Disintegrations per minute

a=         Total number of lymph nodes per animal is 2

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

na=         Not applicable

*=   Significantly different from control group p<0.05

Table 5     Individual Clinical Observations and Mortality Data

Concentration

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 acetone/olive oil 4:1

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

1-5

0

0

0

0

0

0

0

0

0

Test item

5% v/v in acetone/olive oil 4:1

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

2-5

0

0

0

0

0

0

0

0

0

Test item

10% v/v in acetone/olive oil 4:1

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

3-5

0

0

0

0

0

0

0

0

0

Test item

25% v/v in acetone/olive oil 4:1

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

4-5

0

0

0

0

0

0

0

0

0

Positive Control Item

25% v/v in acetone/olive oil 4:1

5-1

0

0

0

0

0

0

0

0

0

5-2

0

0

0

0

0

0

0

0

0

5-3

0

0

0

0

0

0

0

0

0

5-4

0

0

0

0

0

0

0

0

0

5-5

0

0

0

0

0

0

0

0

0


0=    No signs of systemic toxicity

Table 6     Individual Body Weights and Body Weight Change

Concentration

Animal Number

Body Weight (g)

Body Weight Change (g)

Day 1

Day 6

Vehicle acetone/olive oil

 4:1

1-1

21

21

0

1-2

18

19

1

1-3

18

18

0

1-4

18

17

-1

1-5

20

19

-1

Test item

5% v/v in acetone/olive oil 4:1

2-1

18

18

0

2-2

18

18

0

2-3

20

20

0

2-4

18

19

1

2-5

18

17

-1

Test item

10% v/v in acetone/olive oil 4:1

3-1

21

20

-1

3-2

17

18

1

3-3

18

18

0

3.4

19

18

-1

3-5

19

19

0

Test item

25% v/v in acetone/olive oil 4:1

4-1

20

19

-1

4-2

17

16

-1

4-3

18

19

1

4-4

19

18

-1

4-5

18

19

1

Positive Control Item

25% v/v in acetone/olive oil 4:1

5-1

20

20

0

5-2

21

21

0

5-3

19

19

0

5-4

17

18

1

5-5

18

19

1

Ear Thickness Measurements and Mean Ear Thickness Changes are given in Table 7 and Local Skin Irritation in Table 8 (see attachments).

There was no increase in ear thickness (25%) in any of the test or control animals on Days 3 and 6. No signs of irritation were seen in any of the animals throughout the test

            

Ear Weight Measurements are given in Table 9 (see attachments).

There was no increase in ear weight measurements (≥25%) in any of the test or positive control animals on Day 6.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

SUMMARY

INTRODUCTION AND PURPOSE

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010)

Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

United States Environmental Protection Agency Health Effects Test Guidelines OPPTS 870.2600 Skin Sensitization March 2003

The assay has undergone extensive inter‑laboratory validation and has been shown to reliably detect test items that are moderate to strong sensitizers.

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% 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 five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1at concentrations of 25%,10% or 5% v/v. A further group of five animals was treated with acetone/olive oil 4:1alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

 

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:

 

Treatment Group

Concentration

Stimulation Index

Result

Test Item

5% v/v in acetone/olive oil 4:1

0.99

negative

10% v/v in acetone/olive oil 4:1

1.42

negative

25% v/v in acetone/olive oil 4:1

1.51

negative

Positive
Control Item

25% v/v in acetone/olive oil 4:1

4.01

positive

 

Conclusion

The test item was considered to be a non- sensitizer under the conditions of the test.

 

α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

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

The assay has undergone extensive inter‑laboratory validation and has been shown to reliably detect test items that are moderate to strong sensitizers.

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% 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 five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1at concentrations of 25%,10% or 5% v/v. A further group of five animals was treated with acetone/olive oil 4:1alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

 

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:

 

Treatment Group

Concentration

Stimulation Index

Result

Test Item

5% v/v in acetone/olive oil 4:1

0.99

negative

10% v/v in acetone/olive oil 4:1

1.42

negative

25% v/v in acetone/olive oil 4:1

1.51

negative

Positive
Control Item

25% v/v in acetone/olive oil 4:1

4.01

positive

 

Conclusion

The test item was considered to be a non- sensitizer under the conditions of the test.

 

α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1


Migrated from Short description of key information:
The test item was considered to be a non- sensitizer under the conditions of the test.

Justification for selection of skin sensitisation endpoint:
GLP, per OECD method 429 and B42 and Klimisch 1

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item was considered to be a non- sensitizer under the conditions of the test.