Absolute of Cistus ladaniferus (Cistaceae) obtained from labdanum resinoid by ethanol extraction.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June to 02 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 05, 2016/ signed on October 28, 2016)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Absolute of Cistus ladaniferus (Cistaceae) obtained from labdanum resinoid by ethanol extraction.

EC Number:

946-441-0

Molecular formula:

Not applicable

IUPAC Name:

Absolute of Cistus ladaniferus (Cistaceae) obtained from labdanum resinoid by ethanol extraction.

Test material form:

solid

Details on test material:

- Name (as cited in study report): LABDANUM ABSOLUTE / F
- Storage condition of test material: closed containers, room temperature in the dark"

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: at least 15 changes per hour
- Photoperiod: 12 hours continuous light and 12 hours darkness

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50%, 25% or 10% v/v

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Test Item preparation: For the purpose of the study, the test item was freshly prepared, as required, as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- 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 test item at a concentration of 50% v/v 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. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the 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 and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. 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.
- Results: No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay, individual method
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
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.
Local skin irritation was scored daily. The thickness of each ear was measured and recorded pre and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6.
Five days following the first topical application of the test item, vehicle control item or positive control item (Day 6) all mice were injected via the tail vein with 0.25 mL (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.
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, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-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.

Results and discussion

Positive control results:

The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (7.48) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.54

Test group / Remarks:

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

Results 2

Key result

Parameter:

SI

Value:

3.05

Test group / Remarks:

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

Results 3

Key result

Parameter:

SI

Value:

5.35

Test group / Remarks:

50% v/v in acetone/olive oil 4:1

Results 4

Key result

Parameter:

EC3

Remarks:

%

Value:

25

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
See Table 7.4.1/1 below.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 10, 25 and 50% v/v in acetone/olive oil 4:1 were 1.54, 3.05 and 5.35, respectively.

EC3 CALCULATION
EC3 = 2^ {log2(c) + (3-d)/(b-d) x [log2(a) - log2(c)]}
a=10
b=1.54
c=25
d=3.05
EC3 = 25 + [[(3−3.05)/(1.54−3.05)] x (10−25)] = 25
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 25%.

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the test or control animals during the test (See Table 7.4.2 & 7.4.3 below).

BODY WEIGHTS: 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

Table 7.4.1/1: Individual Disintegrations per Minute and Stimulation Index

Treatment Group	Animal Number	dpm/Animal (a)	Mean dpm/Animal (Standard Deviation)	Stimulation Index(b)	Result
Vehicle acetone/olive oil 4:1	1-1	1271.59	1558.64 (±458.45)	na	na
	1-2	2164.52
	1-3	1492.77
	1-4	1010.92
	1-5	1853.41
Test Item 10% v/vin acetone/olive oil 4:1	2-1	3852.08	2406.67 (±961.67)	1.54	Negative
	2-2	1283.76
	2-3	1872.77
	2-4	2368.04
	2-5	2656.72
Test Item 25% v/vin acetone/olive oil 4:1	3-1	5653.60	4748.34** (±1357.71)	3.05	Positive
	3-2	4716.05
	3-3	6087.03
	3-4	4718.41
	3-5	2566.60
Test Item 50% v/vin acetone/olive oil 4:1	4-1	6498.13	8342.72** (±2143.11)	5.35	Positive
	4-2	9562.04
	4-3	10169.34
	4-4	9912.81
	4-5	5571.30
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	8779.42	11659.40** (±4419.51)	7.48	Positive
	5-2	8654.67
	5-3	11254.41
	5-4	19324.29
	5-5	10284.21

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

**=       Significantly different from vehicle control group p<0.01

Table 7.4.1/2: Individual Clinical Observations and Mortality Data

Treatment Group	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/vin 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 25% v/vin 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 50% v/vin 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 7.4.1/3: Local Skin Irritation

Treatment Group	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
Vehicle acetone/olive oil 4:1	1-1	0	0	0	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0	0	0	0
	1-5	0	0	0	0	0	0	0	0	0	0	0	0
Test Item 10% v/vin acetone/olive oil 4:1	2-1	0	0	0	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0	0	0	0
	2-5	0	0	0	0	0	0	0	0	0	0	0	0
Test Item 25% v/vin acetone/olive oil 4:1	3-1	0	0	0	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0	0	0	0
	3-5	0	0	0	0	0	0	0	0	0	0	0	0
Test Item 50% v/vin acetone/olive oil 4:1	4-1	0	0	0	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0	0	0	0
	4-5	0	0	0	0	0	0	0	0	0	0	0	0
Positive Control 15% v/v in acetone/olive oil 4:1	5-1	0	0	0	0	0	0	0	0	0	0	0	0
	5-2	0	0	0	0	0	0	0	0	0	0	0	0
	5-3	0	0	0	0	0	0	0	0	0	0	0	0
	5-4	0	0	0	0	0	0	0	0	0	0	0	0
	5-5	0	0	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the test conditions, test material is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008.

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP. 

Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of 50% 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 five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. 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.

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

Stimulation index for 10%, 25% or 50% v/vin acetone/olive oil 4:1 were 1.54, 3.05 and 5.35, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3value) was calculated to be 25%. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 50%, 25% or 10% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (7.48) when tested at a concentration of 25% v/v inacetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.

 

Under the test conditions, test material is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.