Reaction mass of [3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-6-ol and [3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

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)

Remarks:

inspected June 2015; signature: September 2015

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(1S,2R,5S,7R,8R)-2,6,6,8-tetramethyltricyclo[5.3.1.0^{1,5}]undecan-8-yl acetate

Molecular formula:

C17H28O2

IUPAC Name:

(1S,2R,5S,7R,8R)-2,6,6,8-tetramethyltricyclo[5.3.1.0^{1,5}]undecan-8-yl acetate

Test material form:

liquid

Details on test material:

- Physical state: Liquid.- Storage condition of test material: Approximately 4°C in the dark- Other: Pale yellow

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: recognised animal supplier- Age at study initiation: 8-12 weeks- Weight at study initiation: 15 - 23 grams- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.- Diet: ad libitum - Water: mains tap water ad libitum- Acclimation period: at least 5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 19 - 25- Humidity (%): 30 - 70- Air changes (per hr): 15- Photoperiod: 12 hours light / 12 hours dark

Study design: in vivo (non-LLNA)

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde, tech., 85%

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

- Preliminary test: undiluted (100%)- Main test: undiluted (100%), 50%, 25%, 10% and 1%

No. of animals per dose:

Preliminary test: 1 mouseMain test: 5 mice per dose group

Details on study design:

RANGE FINDING TESTS:Using available information regarding the systemic toxicity/irritancy potential of the test substance, a preliminary screening test was performed using one mouse per test item concentration. The mouse was treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 100% v/v, 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 in the full study report. 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 gauge, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 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 STUDYANIMAL ASSIGNMENT AND TREATMENT- Criteria used to consider a positive response: The test item will be regarded as a sensitiser 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-sensitiser".TREATMENT PREPARATION AND 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 acetone/olive oil 4:1. Additional groups of five animals were treated with the test item at concentrations of 25%, 10% and 1% v/v along with control. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local 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:80μCi/ml, specific activity 2.0 Ci/mmol) 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.- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).- Ear thickness measurements: The thickness of each ear was measured using a gauge, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 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.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 labelled 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 degrees Celsius, 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 (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The 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 a recognised scintillation system.

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:

In a concurrent 'positive control study' performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde (85%) at 25% v/v in acetone/olive oil 4:1. The highest concentration tested showed a Stimulation Index (SI) of 6.83 and met the criteria for a 'positive' result.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: See table below

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See table below

Any other information on results incl. tables

In the preliminary screening test: The preliminary test was conducted at 100%v/v (undiluted) based on available information. 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 the preliminary test concentrations of 100%, 50% and 25%v/v were selected for the main test. Subsequently further test groups 25%, 10% and 1% were conducted in order to more reliably, determine the concentration expected to cause a 3 fold increase in 3HTdR incorporation.

 

In the test, there were no deaths or signs of systemic toxicity. Very slight erythema were noted Days 2 to 5 on both ears at 100%v/v concentration (ear thickness increase: mean = 18.87% day 6. Which was the maximum observed increase). Body weights were comparable to that observed in the corresponding control group over the same period. The and stimulation index (SI) are given in the table for all test item concentrations.

 

Table 1.0 - Stimulation Index during the test

Treatment Group	Concentration	Stimulation Index	Result
Test Item (Initial Test)	25%v/vinacetone/olive oil 4:1	5.26	Positive
	50%v/vin acetone/olive oil 4:1	10.94	Positive
	100%	16.85	Positive
Test Item (Additional Test)	1%v/vin acetone/olive oil 4:1	1.47	Negative
	10%v/vinacetone/olive oil 4:1	1.60	Negative
	25%v/vinacetone/olive oil 4:1	3.87	Positive
Positive Control Item (from Initial Test)	25%v/vinacetone/olive oil 4:1	6.83	Positive

 

Table 2.0 – Radioactive disintegrations per minute (dpm) and stimulation index (SI) (additional test; positive control from initial test)

Treatment Group	Number	dpm / organism #a	Mean dpm /organism (Standard Deviation)	Stimulation Index #b	Result
Vehicleacetone/olive oil 4:1	6-1	854.33	1735.71 (±649.91)	na	na
	6-2	2132.05
	6-3	2069.80
	6-4	1248.78
	6-5	2373.60
Test Item1% v/vinacetone/olive oil 4:1	7-1	977.65	2558.43 (±1399.07)	1.47	Negative
	7-2	2967.07
	7-3	3787.88
	7-4	1178.73
	7-5	3880.80
Test Item10% v/vin acetone/olive oil 4:1	8-1	1785.66	2770.51 (±1647.32)	1.60	Negative
	8-2	3198.73
	8-3	1937.29
	8-4	1460.27
	8-5	5470.59
Test Item25% v/vin acetone/olive oil 4:1	9-1	6904.31	6718.89*** (±1662.67)	3.87	Positive
	9-2	6481.84
	9-3	4799.35
	9-4	6070.48
	9-5	9338.49
Positive Control Item 25% v/v inacetone/olive oil 4:1	5-1	10574.03	12681.51** (±6179.40)	6.83	Positive
	5-1	13031.99
	5-1	3904.01
	5-1	20745.48
	5-1	15152.06

dpm = Disintegrations per minute

#a = Total number of lymph nodes per organism is 2

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

na =Not applicable

*** = Significantly different from control group p<0.001

** = Significantly different from control group p<0.01 (within initial test)

 

Applicant assessment indicates: The initial test indicated a dose-response. An EC3 value (the estimated test substance concentration that will give a SI =3) of 19.0 % was calculated based on linear log extrapolation of EC3 = 19%. To improve the reliability of the estimated EC3 an additional test was conducted. The SI values calculated for the substance concentrations 1%, 10% and 25% in the additional test were 1.47, 1.60 and 3.87 respectively. An EC3 value of 19.0 % was calculated based on linear interpolation of the additional test. The preceding positive control group adequately demonstrated the sensitivity of the test system.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the condition of this study, the test material is considered to sensitising to skin. The concentration of test substance expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 19.0%.

Executive summary:

The study was performed to OECD TG 429 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the test item undiluted (100%) 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 bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. 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 the preliminary test, the concentrations selected for the main test were 0% (control), 25%, 50% and 100% v/v. Groups of five mice were initially treated with the undiluted test item or the test item at concentrations of 100%, 50% or 25% 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 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. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 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 sensitisation. 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. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. 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. Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. 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. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights were comparable to controls. A stimulation index (SI) of more than 3 was noted at the 25, 50 and 100% test concentration groups. The 25% v/v generated a SI = 5.26. Based on linear log extrapolation the EC3 was estimated as 19%. In order to improve the reliability of the the EC3 an additional groups at concentrations of 25%, 10% and 1% and 0% v/v (control) in acetone/olive oil 4:1 was conducted. The test item exhibited a SI = 3.87 at 25%v/v. The EC3 value was calculated by linear interpolation to be 19.0%. The positive control gave a stimulation index > 3.0 thereby demonstrating the sensitivity and reliability of the test system. Accordingly, the test material was considered to be sensitising under the conditions of the test.