Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum 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:

From March 17 to May 13, 2015

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on March 12 to 14, 2014/ signed on May 12, 2014)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction

Molecular formula:

not applicable for UVCB

IUPAC Name:

Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): CISTUS CONCRETE
- Physical state: Dark brown solid at room temperature
- CAS No.: 89997-74-0
- EINECS-No.: 289-711-7
- Analytical purity: 100%UVCB
- Lot/batch No.: 14822 (réf. 101412)
- Production date: 13 May 2013
- Date of Receipt: 11 February 2014
- Expiration date of the lot/batch: 13 May 2015
- Composition: Paraffins, labdanolic derivated
- Storage condition of test material: Room temperature 20 ± 5 °C, keep away from light and humidity

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories BV., Horst, The Netherlands.
- 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 Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From March 17 to May 13, 2015

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 10, 25 and 50 % w/w in acetone/olive oil 4:1
Main test: 2.5, 5 and 10 % w/w in acetone/olive oil 4:1

No. of animals per dose:

Preliminary screening test: One animal/dose
Main test: 5 animals/dose

Details on study design:

RANGE FINDING TESTS:
- Using available information regarding the 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 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 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. 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.

- Irritation: No signs of systemic toxicity or visual local skin irritation were noted. A greater than 25% increase in mean ear thickness was noted at 25 and 50% w/w in acetone/olive oil 4:1. No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted at 10% w/w in acetone/olive oil 4:1. Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in acetone/olive oil 4:1.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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 test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). 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. Five hours later animals were killed by carbon dioxide asphyxiation followed by cervical separation. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) for 18 h at ca. 4 °C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing 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 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).
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

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) were presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Results and discussion

Positive control results:

A group of five animals was treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. With a SI = 13.93, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.75

Test group / Remarks:

at the concnetration of 2.5%

Results 2

Parameter:

SI

Value:

0.66

Test group / Remarks:

at the concentration of 0.66%

Results 3

Key result

Parameter:

SI

Value:

0.98

Test group / Remarks:

at the concentration of 10%

Any other information on results incl. tables

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Bodyweight

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

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

 

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Initial test
Vehicle	1-1	1587.06	2317.87 (±638.87)	NA	NA
	1-2	2650.96
	1-3	1702.12
	1-4	3042.39
	1-5	2606.82
2.5	2-1	1642.11	1730.30 (±385.69)	0.75	Negative
	2-2	1600.90
	2-3	2175.96
	2-4	2032.12
	2-5	1200.40
5	3-1	1219.98	1527.30 (±439.63)	0.66	Negative
	3-2	1132.01
	3-3	1432.20
	3-4	2238.55
	3-5	1613.78
10	4-1	3301.68	2269.87 (±604.29)	0.98	Negative
	4-2	2053.49
	4-3	2289.25
	4-4	1865.95
	4-5	1838.96
Additional test
Vehicle	1-1	1133.87	2745.76 (±1268.51)	NA	NA
	1-2	2662.05
	1-3	2399.08
	1-4	2865.31
	1-5	4668.47
10	2-1	3122.44	2661.90 (±282.53)	0.97	Negative
	2-2	2665.94
	2-3	2595.95
	2-4	2354.49
	2-5	2570.66

 

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) .

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca (CBA/CaOlaHsd) 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 were noted at a 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 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 10%, 5% or 2.5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. Due to the discrepancy in the weight of the test item used on Day 3 of the main test, an additional group of five animals was treated with the test item at a concentration of 10% w/w in acetone/olive oil 4:1 to confirm the results of the maximum suitable concentration. An additional group of five animals was treated with acetone/olive oil 4:1 alone.

 

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	Mean dpm/animal	Stimulation Index	Result
Initial test
Vehicle	2317.87 ± 638.87	NA	NA
2.55	1730.30 ± 385.69	0.75	Negative
5	1527.30 ± 439.63	0.66	Negative
10	2269.87 ± 604.29	0.98	Negative
Additional test
Vehicle	2745.76 ± 1268.51	NA	NA
10	2661.90 ± 282.53	0.97	Negative

 

 

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 13.93, when tested at 25 % v/v. The test system was therefore considered to be valid.

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) .

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