[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 18 April 2016 and 05 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

450-320-3

EC Name:

-

Molecular formula:

C15H28O

IUPAC Name:

(3S,6E)- 3,7,11-trimethyldodeca-6,10-dien-1-ol

Test material form:

liquid

Details on test material:

Identification: l-Dihydrofarnesol
Batch: 1203158
Chemical Name: (3S,6E)-3,7,11-Trimethyldodeca-6,10-dien-1-ol
CAS Number: 27745-36-4 (3S,6E), 51411-24-6 (planar)
CAS Name: 6,10-Dodecadien-1-ol, 3,7,11-trimethyl-, (3S,6E)-
Physical state/Appearance: colorless to pale yellow liquid
Purity: 97.5%
Expiry Date: not supplied
Storage Conditions: room temperature in the dark

Specific details on test material used for the study:

Identification: l-Dihydrofarnesol
Batch: 1203158
Chemical Name: (3S,6E)-3,7,11-Trimethyldodeca-6,10-dien-1-ol
CAS Number: 27745-36-4 (3S,6E), 51411-24-6 (planar)
CAS Name: 6,10-Dodecadien-1-ol, 3,7,11-trimethyl-, (3S,6E)-
Physical state/Appearance: colorless to pale yellow liquid
Purity: 97.5%
Expiry Date: not supplied
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaCrl)

Sex:

female

Details on test animals and environmental conditions:

Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 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 8 to 12 weeks old.
Animal Care and Husbandry
The animals were 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 Envigo RMS (UK) Limited, 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 at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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.

Study design: in vivo (LLNA)

Vehicle:

other: diethyl phthalate/ethanol 3:1

Concentration:

Preliminary screening test: 100%
Main test: 100%, 50% & 25%

No. of animals per dose:

Preliminary screening test: 1
Main test: 5 per dose

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item 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. Local skin irritation was scored daily according to the scale included as Annex 4. Any clinical signs of toxicity, if present, were also recorded. The body weight 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 undiluted test item or the test item at concentrations of 50% or 25% v/v in diethyl phthalate/ethanol 3:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. 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.
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).
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 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by  scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 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:

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. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

Positive control results:

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
diethyl phthalate/ethanol 3:1 Stimulation Index Result
25 5.52 Positive

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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.36

Test group / Remarks:

25%

Results 2

Parameter:

SI

Value:

10.36

Test group / Remarks:

50%

Results 3

Parameter:

SI

Value:

13.71

Test group / Remarks:

100%

Results 4

Parameter:

EC3

Value:

21.4

Cellular proliferation data / Observations:

Clinical Observations and Mortality Data
Individual clinical observations and mortality data for test and control animals are given inAppendix 5.
Slight redness of the ears and neck was noted, post dose on Day 3 and on Days 4 to 6, in animals treated with the undiluted test item. 
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Body Weight
Individual body weights and body weight change for test and control animals are given inAppendix 6.
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Preliminary Screening Test

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

Very slight erythema was noted on both ears on Days 3 and 4. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in diethyl phthalate/ethanol 3:1 were selected for the main test.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Appendix 4.

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 (%v/v) in diethyl phthalate/ethanol 3:1	Stimulation Index	Result
25	4.36	Positive
50	10.36	Positive
100	13.71	Positive

Calculation of EC₃Value

EC₃= 2^ {log₂(c) + (3-d)/(b-d) x [log₂(a)-log₂(c)]}

a	=	50
b	=	10.36
c	=	25
d	=	4.36
EC3= 2^ {log2(25) + (3-4.36)/(10.36-4.36) x [log2 (50)-log2(25)]} =21.4

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (extrapolated EC₃value) was calculated to be 21.4%.

Appendix1     Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Concentration	Animal Number	Body Weight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
100%	S-1	20.5	20.9	0	0	0	0	0	0	0	0	0

0=    No signs of systemic toxicity

Appendix2     Local Skin Irritation – Preliminary Screening Test

Concentration

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

100%

S-1

0

0

0

0

1

1

1

1

0

0

0

0

Appendix3     Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
100%	S-1	0.23	0.23	0.26	0.25	0.25	0.25
overall mean (mm)		0.230		0.255		0.250
overall mean ear thickness change (%)		na		10.870		8.696

na=  Not applicable

Appendix4     Individual Disintegrations per Minute and Stimulation Index

Concentration (% v/v) in diethyl phthalate/ethanol 3:1	Animal Number	dpm/ Animala	Mean dpm/Animal (Standard Deviation)	Stimulation Indexb	Result
Vehicle	1-1	523.62	634.70 (±85.48)	na	na
	1-2	593.48
	1-3	692.71
	1-4	621.10
	1-5	742.58
25	2-1	2695.30	2768.14** (±560.43)	4.36	Positive
	2-2	3512.29
	2-3	3147.15
	2-4	2274.79
	2-5	2211.18
50	3-1	5025.20	6573.41** (±1511.53)	10.36	Positive
	3-2	5417.10
	3-3	8548.56
	3-4	6151.43
	3-5	7724.78
100	4-1	9345.33	8704.87** (±2401.76)	13.71	Positive
	4-2	7560.30
	4-3	8165.43
	4-4	12429.62
	4-5	6023.69

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<0.01

Appendix5     Individual Clinical Observations and Mortality Data

Concentration (% v/v) in diethyl phthalate/ethanol 3:1		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
		1-5		0		0		0		0		0		0		0		0		0
25		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
50		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
100		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*

0=    No signs of systemic toxicity

* =   Slight redness of the ears and neck

Appendix6     Individual Body Weights and Body Weight Change

Concentration (% v/v) in diethyl phthalate/ethanol 3:1	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	20.8	21.8	1.0
	1-2	18.8	18.7	-0.1
	1-3	20.3	20.7	0.4
	1-4	19.4	20.1	0.7
	1-5	20.0	20.4	0.4
25	2-1	21.1	21.2	0.1
	2-2	18.8	19.7	0.9
	2-3	19.8	20.1	0.3
	2-4	18.8	19.3	0.5
	2-5	17.7	18.8	1.1
50	3-1	19.6	20.6	1.0
	3-2	17.8	18.0	0.2
	3-3	18.8	19.2	0.4
	3-4	19.5	19.5	0.0
	3-5	18.5	18.4	-0.1
100	4-1	18.7	17.8	-0.9
	4-2	15.0	15.1	0.1
	4-3	18.7	18.7	0.0
	4-4	18.9	19.3	0.4
	4-5	21.1	20.9	-0.2

Applicant's summary and conclusion

Interpretation of results:

other: The test item was considered to be a sensitizer under the conditions of the test.

Conclusions:

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

Executive 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.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, 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 undiluted test item or the test item as a solution in diethyl phthalate/ethanol 3:1 at concentrations of 50% or 25% v/v. A further group offive animals was treated with diethyl phthalate/ethanol 3:1 alone.

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 (%v/v) in diethyl phthalate/ethanol 3:1	Stimulation Index	Result
25	4.36	Positive
50	10.36	Positive
100	13.71	Positive

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (extrapolated EC₃value) was calculated to be 21.4%.

Conclusion

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