Registration Dossier

Administrative data

Description of key information

In a well conducted guideline LLNA study in mice (Harlan Laboratories, 2014a) there was no evidence that farnesane induced skin sensitisation.

The lack of skin sensitisation potential was supported by results of 3 human repeat insult patch tests in which over 300 subjects were exposed dermally to concentrations of farnesane between 20 and 80%. It is concluded that farnesane does not have the potential to induce skin sensitisation.

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:
2014
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)
Remarks:
Appendix 4
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 group 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 temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70%, respectively. 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:
Preliminary Screening Test
50%, 25% and 40% v/v in acetone/olive oil 4:1

Main Test
Each group was exposed to concentrations of 40%, 20% or 10% 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 three 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%, 25% and 40% 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 3. 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 40%, 20% or 10% 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 40% 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 (post dose) 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 study 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 25% 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 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
25 7.96 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Value:
0.88
Test group / Remarks:
10% v/v in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
1.29
Test group / Remarks:
20% v/v in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
1.04
Test group / Remarks:
40% v/v in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
7.96
Test group / Remarks:
Positive Control Item 25% v/v in acetone/olive oil 4:1
Remarks on result:
other: Positive
Key result
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 4 (Any other information).
Key result
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the the test material at concentrations of 40%, 20% and 10% v/v in acetone/olive oil 4:1. The stimulation index (SI) results are given in Table 2.

Clinical observations, body weight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

The mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1 showed a greater than 25% increase in mean ear thickness on Day 3. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the animals treated at concentrations of 40% and 25% v/v in acetone/olive oil 4:1. 

 

Based on this information the dose levels selected for the main test were 40%, 20% and 10% v/v in acetone/olive oil 4:1.

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 vehicle control group animals over the same period.

Ear Thickness Measurements and Mean Ear Thickness Changes are given in Table 7 and Local Skin Irritation in Table 8. 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 anyof the test or vehicle control animals throughout the test. Very slight irritation was noted in all the positive control animals on Days 1 to 3.

Ear Weight Measurements are given in Table 9.

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

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

3114.65

3404.89
(±599.03)

na

na

1-2

4269.29

1-3

3679.70

1-4

2690.58

1-5

3270.21

Test Item

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

2-1

3596.13

2982.33
(±839.48)

0.88

Negative

2-2

1623.30

2-3

2905.28

2-4

3747.88

2-5

3039.08

Test Item

20% v/v in acetone/olive oil 4:1

3-1

3925.89

4379.98
(±689.38)

1.29

Negative

3-2

4304.52

3-3

4981.94

3-4

5157.43

3-5

3530.10

Test Item

40% v/v in acetone/olive oil 4:1

4-1

4038.92

3542.80
(±1055.93)

1.04

Negative

4-2

5074.29

4-3

3235.71

4-4

3065.87

4-5

2299.19

Positive Control Item

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

5-1

26297.31

27104.81***
(±6209.10)

7.96

Positive

5-2

25972.97

5-3

37776.86

5-4

23095.96

5-5

22380.95

 

The results of the statistical analysis of the data indicated there was no significant difference between the vehicle 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 vehicle control group p<0.001

Table5     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

10% 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

20% 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

40% 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

Table6     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

18

18

0

1-2

19

19

0

1-3

17

18

1

1-4

20

20

0

1-5

16

17

1

Test item

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

2-1

18

18

0

2-2

20

20

0

2-3

18

20

2

2-4

18

18

0

2-5

18

19

1

Test item

20% v/v in acetone/olive oil 4:1

3-1

20

21

1

3-2

19

19

0

3-3

18

18

0

3-4

18

19

1

3-5

16

17

1

Test item

40% v/v in acetone/olive oil 4:1

4-1

19

19

0

4-2

19

19

0

4-3

18

19

1

4-4

19

19

0

4-5

20

20

0

Positive Control Item

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

5-1

19

19

0

5-2

20

22

2

5-3

17

18

1

5-4

20

19

-1

5-5

20

21

1

Interpretation of results:
other: Not sensitising
Remarks:
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

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 LymphNodeAssay" (adopted 22 July 2010)

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

  

The assay has undergone extensive inter‑laboratory validation and has been shown to reliably detect test items that are moderate to strong sensitizers. The strain of mouse used in these laboratories has been shown to produce satisfactory responses using known sensitizers and non‑sensitizers during the in‑house validation. The results of routine positive control studies areshown in Appendix 1. The results of the study are believed to be of value in predicting the sensitization potential of the test item to man.

      

Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse, and are specified in the appropriate test guidelines.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of 40% v/v, this concentration was selected as the highest dose investigated inthe main test of the Local Lymph Node Assay. Three groups, each of five animals, were treatedwith 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1at concentrations of 40%,20% or10% v/v. A further group of five animals was treated withacetone/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

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

0.88

Negative

20% v/v in acetone/olive oil 4:1

1.29

Negative

40% v/v in acetone/olive oil 4:1

1.04

Negative

Positive
Control Item

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

7.96

Positive

 

Conclusion

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

 The positive control material 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:

Farnesane showed no activity in a well conducted mouse local lymph node assay (Harlan laboratories, 2014a).

In an independent human repeat insult patch test by Harrison Research labs, a panel of over 100 subjects was commissioned for testing initially with 80% farnesane in mineral oil under semi-occlusive conditions. The application regime consisted of an induction period of repeat applications every 48 hours with patch removal after 24 hours, a rest period of two weeks and then a final Challenge application and analysis. After the first 48 hours, several subjects showed mild erythema and one subject showed edema which prompted reduction of the test concentration to 55%. Subsequent applications of both substances at this concentration produced no adverse effects. The results of this study indicated that farnesane does not elicit an allergic response.

While some irritation was observed in this study, it is important to note that the testing was conducted at high, exaggerated and localized concentrations which are above typical usage concentrations. Refer to the irritation endpoint summary and a review of the extensive studies indicating that farnesane is not classifiable as a skin irritant.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

.

Justification for classification or non-classification

Farnesane showed no activity in a well conducted mouse local lymph node assay. This lack of activity is supported by 3 human repeat insult patch tests - see below.

Human Clinical Controlled Studies to Assess Potential for Sensitization of Farnesane

Three successive HRIPTs on farnesane were carried out at well- established testing facilities that have routinely conducted human clinical allergy testing for many years. The testing facilities used were:

·        Product Investigations Inc. (PII) located in either Conshohocken, PA or Modesto, CA. under the supervision of Dr. Morris Shelanski

·        Harrison Research Labs (HRL) located in Union, NJ. under the supervision of Lynne B Harrison, PhD.

Three separate panels across two separate locations were assembled for HRIPTs with over 100 subjects per panel. The panels were composed of a wide cross-section of healthy individuals, with equal gender representation, who had been screened for health history and trained on the patch testing process.  Prior to initiation of the test at Harrison Research Lab, a preliminary safety review of farnesane was carried out by an Internal Review Board (IRB) and farnesane was cleared for testing under the established protocol. The testing facilities employed established and recognised methods for patch testing and scoring. A certified dermatologist was present to review results, as needed throughout the studies, and during the challenge phase.  

Supporting studies

At PII, two HRIPTs were carried out with separate panels of over 100 subjects at two different concentrations. One panel examined the effects of a 20% concentration of farnesane and a subsequent panel was initiated to examine the effects of applications of undiluted (100%) material. The testing regime for studies consisted of an induction period of three weeks where repeat applications of ~200 µL of the substance in mineral oil or undiluted were applied to the upper arms of subjects under occlusive conditions. Every 24 hours, subjects were instructed to return to the clinic for removal of the patch, grading of the response and repatching with a fresh patch. Applications were made every day except over the weekend. A one week rest period followed these applications and then applications were resumed on week five as part of the Challenge Phase.

Results of the HRIPT with 20% farnesane in mineral oil showed no indication of either irritancy or sensitization. Results with 100% farnesane were more complex. After 24 hours at 100% farnesane approximately 30% of the subjects were showing irritation, which prompted a change in application procedure. Instead of covering the application site, open applications at 100% were made for one week. Under these conditions the skin tolerated farnesane well and no evidence of irritation was noted. For the 3rd week, applications were made at reduced concentrations under semi-occlusive conditions and under these conditions minor irritation was noted. Following the rest period the skin was challenged with a 60% concentration of farnesane under occlusive condition which ultimately was raised to 80% by the weeks end with no evidence of sensitization and very only a mild irritant response. The results of this study indicate that farnesane does not elicit an allergic response.

Key Study

In a separate independent study by Harrison Research labs, another panel of over 100 subjects was commissioned initially with 80% farnesane in mineral oil under semi-occlusive conditions. The application regime consisted of an induction period of repeat applications every 48 hours with patch removal after 24 hours, a rest period of two weeks and then a final Challenge application and analysis. After the first 48 hours, several subjects showed mild erythema and one subject showed edema which prompted reduction of the test concentration to 55%. Subsequent applications at this concentration produced no adverse effects. The results of this study indicated that farnesane does not elicit an allergic response.

 

While some irritation was observed in some of these studies, it is important to note that the testing was conducted at high, exaggerated and localized concentrations which are above typical usage concentrations. Refer to the Irritation endpoint summary and a review of the extensive studies indicating that farnesane is not classifiable as a skin irritant.

 

In conclusion, farnesane was tested in over 300 human panelists and was found to not induce skin sensitization.